Antidotes for factor Xa inhibitors and methods of using the same

ABSTRACT

The present invention relates antidotes to anticoagulants targeting factor Xa. The antidotes are factor X and factor Xa protein derivatives that bind to the factor Xa inhibitors thereby substantially neutralizing them but do not assemble into the prothrombinase complex. The derivatives describe herein lack or have reduced intrinsic coagulant activity. Disclosed herein are methods of reversing anticoagulation, stopping or preventing bleeding in a patient that is currently undergoing anticoagulant therapy with a factor Xa inhibitor.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claim benefit under 35 U.S.C. §119(e) of U.S.Provisional Patent Application 61/164/886, filed Mar. 30, 2009. Thisapplication also is a continuation-in-part of U.S. Ser. No. 12,239,6515,file on Sep. 26, 2008, which application claimed the benefit under 35U.S.C. §119(e) of U.S. Provisional Patent Application Ser. Nos.60/976,343 and 61/090,651, file Sep. 28, 2007 and Aug. 20, 2008,respectively. All of the above-listed patent applications are herebyincorporated by reference in their entirety.

FIELD OF INVENTION

The present invention relates to the use of factor X (fX) and factor Xa(fXa) derivatives having reduced or lacking intrinsic procoagulantactivity but are also capable of binding and/or neutralizing fXainhibitors thereby acting as antidotes to anticoagulants targeting fXa.

BACKGROUND OF THE INVENTION

Anticoagulants serve a need in the marketplace in treatment orprevention of undesired thrombosis in patients with a tendency to formblood clots, such as, for example, those patients having clottingdisorders, confined to periods of immobility or undergoing medicalsurgeries. One of the major limitations of anticoagulant therapy,however, is the bleeding risk associated with the treatments, andlimitations on the ability to rapidly reverse the anticoagulant activityin case of overdosing or if an urgent surgical procedure is required.Thus, specific and effective antidotes to all forms of anticoagulanttherapy are highly desirable. For safety considerations, it is alsoadvantageous to have an anticoagulant-antidote pair in the developmentof new anticoagulant drugs.

Currently available anticoagulant-antidote pairs forover-anticoagulation are heparin-protamine and warfarin-vitamin K. Freshfrozen plasma and recombinant factor VIIa (rfVIIa) have also been usedas non-specific antidotes in patients under low molecular weight heparintreatment, suffering from major trauma or severe hemorrhage. (Lauritzen,B. et al, Blood, 2005, 607A-608A.) Also reported are protamine fragments(U.S. Pat. No. 6,624,141) and small synthetic peptides (U.S. Pat. No.6,200,955) as heparin or low molecular weight heparin antidotes; andthrombin muteins (U.S. Pat. No. 6,060,300) as antidotes for thrombininhibitors. Prothrombin intermediates and derivatives have been reportedas antidotes to hirudin and synthetic thrombin inhibitors (U.S. Pat.Nos. 5,817,309 and 6,086,871).

One promising form of anticoagulant therapy targets factor Xa (fXa), andin fact, several direct fXa inhibitors are currently in different stagesof clinical development for use in anticoagulant therapy. One direct fXainhibitor Xarelto™ (rivaraoxaban) has been approved for clinical use inthe European Union and Canada for the prevention of venousthromboembolism in orthopedic surgery patients. Many of these are smallmolecules. While these new fXa inhibitors show promise for treatment,specific and effective antidotes are still needed. In cases ofover-anticoagulation or requirement for surgery in patients treated withthese fXa inhibitors, an agent may be required to substantiallyneutralize the administered fXa inhibitor or inhibitors and restorenormal hemostasis.

Currently available agents, such as recombinant factor VIIa (rfVIIa),are mechanistically limited and not specific for reversal of fXainhibitors and thus improved options for the clinician are highlydesirable. In human studies, rfVIIa has been used to reverse the effectof indirect antithrombin III dependent fXa inhibitors such asfondaparinux and idraparinux (Bijsterveld, N R et al, Circulation, 2002,106:2550-2554; Bijsterveld, N R et al, British J. of Haematology,2004(124): 653-658). The mechanism of action of factor VIIa (fVIIa) isto act with tissue factor to convert factor X (fX) present in bloodcirculation to fXa to restore normal hemostasis in patients. This modeof action necessarily dictates that the highest potential concentrationof fXa that could be attained to neutralize active site directed fXainhibitors is limited by the circulating plasma concentration of fX.Since the circulating plasma concentration of fX is 150 nanomolar(“nM”), the maximal amount of fXa produced by this mode would be 150 nM.Thus, the potential of using rfVIIa to reverse the effect of direct fXainhibitors is mechanistically limited. Reported therapeuticconcentrations of small molecule fXa inhibitors such as rivaroxaban havebeen higher (approximately 600 nM, Kubitza D, et al, Eur. J. Clin.Pharmacol., 2005, 61:873-880) than the potential amount of fXa generatedby rfVIIa. Use of rfVIIa for reversal of therapeutic or supratherapeuticlevels of anticoagulation by fXa inhibitor would therefore provideinadequate levels of efficacy. As shown in FIG. 4, using rfVIIa haslimited effect in neutralizing the anticoagulant activity of a factor Xainhibitor betrixaban (described below). Recombinant fVIIa showed a doseresponsive antidote activity from 50 nM to 100 nM, but the effectleveled off between 100 nM to 200 nM, indicating that its antidoteeffect is limited by factors other than its concentration. In all of therfVIIa concentrations tested, betrixaban still showed a dose responsiveinhibition of fXa, up to about 75% inhibition at a concentration of 250nM. This observation is consistent with fVIIa's proposed mechanism ofaction. This is also supported by studies showing that rfVIIa did notcompletely reverse the inhibitory effect of fondaparinux on theparameters of thrombin generation and prothrombin activation.(Gerotiafas, G T, et al, Thrombosis & Haemostasis 2204(91):531-537).

Exogenous active fXa cannot be administered directly to a subject in away similar to rfVIIa. Unlike rfVIIa, which has very low procoagulantactivity in the absence of its cofactor tissue factor, native fXa is apotent enzyme and has a potential risk of causing thrombosis. Thus, theuse of either rfVIIa or active fXa as an antidote to a fXa anticoagulanttherapy has disadvantages.

Thus, there is a need for improved antidote agents that do not causeundesired thrombosis and that are effective in substantiallyneutralizing the anticoagulant activity of a fXa inhibitor in the eventof an overdose of the fXa inhibitor or in the event that normalhemostasis needs to be restored to prevent or stop bleeding.

Any and all publications, patents, patent applications mentioned hereinare hereby incorporated by reference in their entirety.

SUMMARY OF THE INVENTION

It has now been discovered that administration of modified derivativesof fX proteins are useful as antidotes to anticoagulants targeting fXa.The modified derivatives of fX proteins do not compete with fXa inassembling into the prothrombinase complex, but instead bind and/orsubstantially neutralize the anticoagulants, such as fXa inhibitors. Thederivatives useful as antidotes are modified to reduce or removeintrinsic procoagulant and anticoagulant activities, while retaining theability to bind to the inhibitors. It is contemplated that thederivatives of the invention may include modifying the active site andchanging or removing the entire Gla domain from fXa. It is furthercontemplated that modification of the Gla domain reduces or removes theanticoagulant effect of the fX derivative on normal hemostasis becausean active site modified full length fXa is known to be an anticoagulant.

Thus, in one embodiment, the invention is directed to an isolatedpolypeptide factor X derivative comprising a modified active site and amodified or deleted Gla domain. It is contemplated that the derivativebinds to a factor Xa inhibitor and does not assemble into aprothrombinase complex.

Additional modifications to the derivative include, but are not limitedto the following: all or part of the activation peptide is deleted at anN-terminus of a heavy chain; the derivative has an amino acidmodification which prevents cleavage of a β-peptide; a β-peptide isdeleted; modification of an EGF domain; the derivative comprises asignal peptide; there is a mutation in the autolysis loop of the factorXa heavy chain; and/or the linker that connects the light chain to theactivation peptide is modified.

In one embodiment, the derivative maintains the structuralcharacteristics necessary for binding the anticoagulant (or fXainhibitor) targeting fXa. By the derivative binding, either directly orindirectly, to the inhibitor, the inhibitor is substantiallyneutralized.

In one aspect, the invention provides a method of neutralizing orreversing anticoagulation in a subject undergoing anticoagulant therapywith a factor Xa inhibitor who is in need of cessation ofanticoagulation or suffering from bleeding. The method comprisesadministering to the subject an effective amount of a factor Xderivative that binds to the factor Xa inhibitor but does not assembleinto the prothrombinase complex. In one embodiment, the derivative haseither reduced or no procoagulant activity. The derivative of theinvention selectively binds and inhibits an exogenously administeredfactor Xa inhibitor thereby substantially neutralizing the anticoagulantactivity of a fXa inhibitor. This method contemplates both in vitro andin vivo methods. It is further contemplated that the methods of theinvention are also directed to preventing or reducing bleeding in asubject undergoing anticoagulant therapy with a factor Xa inhibitor.

It is to be understood that the derivatives contemplated by thisinvention are not plasma derived factor VIIa, recombinant factor VIIa,fresh frozen plasma, prothrombin complex concentrates, or whole blood.

One aspect of the present invention is the use of the factor Xderivatives and compositions containing the same to treat patients whohave received or are receiving over-anticoagulation therapy with afactor Xa inhibitor or patients who had previously been administered afactor Xa inhibitor and is then in need of hemostasis, such as requiredby elective or emergency surgery. In one aspect, the fX derivatives aredistinguished from naturally occurring fX or fXa in that they havereduced or removed intrinsic procoagulant activity and will notinterfere with physiological fXa function in hemostasis, while stillcapable of binding and substantially neutralizing fXa inhibitors.

In another aspect, the modified factor X protein is co-administered withan agent capable of extending the plasma half life (or circulating halflife) of the factor X derivative. In yet another aspect, the antidote isconjugated with a moiety to extend its plasma half-life.

Also provided are pharmaceutical compositions that contain the factor Xderivative that binds (and/or substantially neutralizes) the factor Xainhibitor but does not assemble into the prothrombinase complex. Thepharmaceutical composition optionally comprises a pharmaceuticallyacceptable carrier.

In another aspect, this invention provides a kit comprising a fXinhibitor for anticoagulant use and a fXa inhibitor antidote (or factorXa derivative) for use when substantial neutralization of the fXainhibitor's anticoagulant activity is needed.

One embodiment of the invention is directed to an isolated polypeptidecomprising the amino acid sequence of SEQ ID NO. 12, 13, 15, 19, 20, 21,22, 23, 24, or 25 or a polypeptide having at least 80% homology to 12,13, 15, 19, 20, 21, 22, 23, 24, or 25. The polypeptides encompassed bythe invention include both the single chain polypeptide (i.e., beforethe polypeptide is excreted from the cell) as well as the two-chainpolypeptide (i.e., after cleavage at the linker after it is secretedfrom the cell).

It is contemplated that the polypeptides of SEQ ID NOs. 19 to 25 improveoverall expression of the functional antidote and/or reduceheterogeneity at the C-terminus of the heavy chain.

Also provided is a pharmaceutical composition comprising a carrier and apolypeptide just described.

Also provided is a polynucleotide encoding for a polypeptide justdescribed.

Further provided herein is a peptide conjugate comprising a carriercovalently or non-covalently linked to a polypeptide just described. Thecarrier can be a liposome, a micelle, a pharmaceutically acceptablepolymer, or a pharmaceutically acceptable carrier.

This invention is also directed to an antibody that binds a polypeptidejust described. The antibody is a polyclonal antibody, a monoclonalantibody, a chimeric antibody or a humanized antibody. Also provided isa biologically active fragment of the antibody. The antibody may bedetectably labeled. The antibody (or antibody fragment) is also providedas part of a composition further comprising a carrier.

In one embodiment there is provided an antibody-peptide complexcomprising the antibody of invention and a polypeptide that specificallybinds to the antibody. The polypeptide is the polypeptide against whichthe antibody is raised. The antibody may be a polyclonal antibody, amonoclonal antibody, a chimeric antibody or a humanized antibody.

In another embodiment, there is provided a method for preparing apolypeptide of the invention comprising expressing a polynucleotideencoding the polypeptide in a prokaryotic or eukaryotic host cell. Inone embodiment, the host cell is a eukaryotic cell and in particular isa Chinese hamster ovary cell. In another embodiment, the polypeptide isisolated.

In yet another embodiment, there is provided an isolated prokaryotic oreukaryotic host cell comprising a polynucleotide encoding polypeptide ofthe invention. In one embodiment, the host cell is in a compositionfurther comprising a carrier.

In still yet another embodiment, there is provided a method ofpreventing or reducing bleeding in a subject undergoing anticoagulanttherapy with a factor Xa inhibitor comprising administering to thesubject an effective amount of a composition comprising a polypeptide ofthe invention and a carrier. Further provided is a method of selectivelybinding and inhibiting an exogenously administered factor Xa inhibitorin a subject undergoing anticoagulant therapy comprising administeringto the subject an effective amount of a composition just described.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows schematically the domain structure of human factor X (SEQID NO. 1) shown in Table 1 as reported in Leytus et al, Biochem., 1986,25, 5098-5102. SEQ ID NO. 1 is the amino acid sequence of human fX codedby the nucleotide sequence of human fX (SEQ ID NO. 2) as shown in Table2 known in the prior art. For example, the translated amino acidsequence is reported in Leytus et al, Biochem., 1986, 25, 5098-5102 andcan be found in GenBank, with accession number “NM_(—)000504”. The aminoacid numbering in this sequence is based on fX sequence. Human fXprecursor (SEQ ID NO. 1) contains a prepro-leader sequence (amino acids1 to 40 of SEQ ID NO. 1) followed by sequences corresponding to the fXlight chain (LC) (amino acids 4) to 179 of SEQ ID NO. 1), the RKRtriplet (amino acids 180 to 182 of SEQ ID NO. 1) which is removed duringfX secretion, and the fX heavy chain (amino acids 183 to 488 of SEQ IDNO. 1) containing the activation peptide (AP) (amino acids 183 to 234 ofSEQ ID NO. 1) and the catalytic domain (amino acids 235 to 488 of SEQ IDNO. 1).

FIG. 2 (SEQ ID NO. 3) shows the amino acid sequence of mature humanfactor X. The amino acid numbering in this figure is based on mature fXsequence starting from the N-terminal of fX light chain. Factor Xcirculates in plasma as a two-chain molecule linked by a disulfide bond.The light chain (LC) has 139 amino acid (amino acids 41 through 179 ofSEQ ID NO. 1) residues and contains the γ-carboxyglutamic acid(Gla)-rich domain (amino acids 1-45 of SEQ ID NO. 3), including a shortaromatic stack (AS) (amino acids 40-45 of SEQ ID NO. 3), followed by twoepidermal growth factor (EGF)-like domains (EGF1: amino acids 46-84,EGF2: amino acids 85-128 of SEQ ID NO. 3). The heavy chain (HC) has 306amino acids and contains a 52 amino acids activation peptide (AP: aminoacids 143-194 of SEQ ID NO. 3) followed by the catalytic domain (aminoacids 195-448 of SEQ ID NO. 3). The catalytic triad equivalents toH57-D102-S195 in chymotrypsin numbering are located at His236, Asp282,and Ser379 in fX sequence and are underlined (amino acids 236, 282 and379 of SEQ ID NO. 3).

FIG. 3 shows schematically the domain structure of mature human factor Xshown in FIG. 2. The amino acid numbering in this figure is based onmature fX sequence. The cleavage sites for chymotrypsin digestion toremove the Gla-domain containing fragment (amino acid 1-44 of SEQ ID NO.3) and fX activation to remove the activation peptide are highlighted.Chymotrypic digestion of fXa results in a Gla-domainless fXa lacking the1-44 amino acid residues (SEQ ID NO. 4).

FIG. 4 shows the effect of varying concentrations of rfVIIa (referred toas VIIa in the figure) in the presence of tissue factor (TF) on theanticoagulant, activity of a fXa inhibitor betrixaban (described below)in a thrombin generation (expressed as relative fluorescence units (RFU)assay as described in Example 2). The data show that a combination ofrfVIIa and tissue factor was unable to completely neutralize theanticoagulant activity of a fXa inhibitor, betrixaban, in concentrationsup to 200 nM of rVIIa.

FIG. 5 shows that anhydro-fXa with its Gla-domain intact reverses fXainhibition by betrixaban in a purified system containing active fXa andbetrixaban (open circle), while anhydro-fXa alone has negligibleprocoagulant activity (open triangle) compared with active fXa. FXachromogenic activity was normalized to active fXa in the absence of anyinhibitor (open square). This is more thoroughly described in Example 4.The data show that anhydro-fXa is inactive toward fXa substrate yetretains the fXa inhibitor binding ability.

FIG. 6 shows that the anhydro-fXa with intact Gla domain in FIG. 5 is apotent inhibitor in plasma thrombin generation (expressed as relativefluorescence units (RFU)) assay (as described in Example 2). It almostcompletely inhibited thrombin generation at about 115 nM. The data showthat anhydro-fXa without modification of the Gla-domain is not suitablefor use as a fXa inhibitor antidote.

FIG. 7 shows the comparison of the clotting activity of active fXa in a96-well plate format before chymotrypsin digestion, and after 15 minutesand 30 minutes of chymotrypsin digestion. As shown in this figure,clotting time (change of OD405) was significantly delayed after the fXahad been digested by chymotrypsin for 15 minutes and no clotting wasobserved for up to 20 minutes when the fXa was digested for 30 minutes.This result was also used to establish conditions for chymotrypsindigestion of anhydro-fXa because it has no activity that can bemonitored during digestion. This is more thoroughly described in Example3.

FIG. 8 shows the binding affinity of des-Gla anhydro-fXa to a factor Xainhibitor betrixaban as described in Example 4. The data show thatdes-Gla anhydro-fXa, prepared by chymotryptic digestion of anhydro-fXato remove the Gla-domain containing fragment (residues 1-44), is able tobind betrixaban with similar affinity as native fXa (fXa: Ki=0.12 nM,des-Gla anhydro-fXa: Kd=0.32 nM).

FIG. 9 shows reversal of the anticoagulant activity of varyingconcentrations of betrixaban by addition of a concentrate of 680 nM ofthe antidote (des-Gla anhydro-fXa) in a thrombin generation assay ofExample 2. At the concentration of 680 nM, des-Gla anhydro-fXa was ableto produce substantially complete restoration of fXa activity.

FIG. 10 shows reversal of the anticoagulant activity of 250 nM ofbetrixaban by varying concentrations of the antidote (des-Glaanhydro-fXa) in clotting prolongation assays using aPTT reagent in a96-well plate format (as described in Example 3). The data show thatclotting time was comparable to that of control platelet poor plasma(PPP) when about 608 nM of the antidote was used to neutralize 250 nM ofthe fXa inhibitor betrixaban.

FIG. 11 shows the effect on the anticoagulant activity of enoxaparin(0.3125-1.25 U/mL) by 563 nM of the antidote (des-Gla anhydro-fXa) inclotting prolongation assays using aPTT reagent in a 96-well plateformat, expressed as fold changes after normalization. The assayprotocol is described in Example 3. The data show that addition of 563nM of the antidote significantly neutralized the activity of a lowmolecular weight heparin enoxaparin.

FIG. 12 shows the effect of the antidote, des-Gla anhydro-fXa, on theactivity of thrombin (5 nM) and its inhibition by 50 nM of argatroban, aspecific thrombin inhibitor, in a chromogenic assay. The antidote of fXainhibitor does not detectably affect either thrombin activity or itsinhibition by the specific inhibitor argatroban at concentrations up to538 nM. This is more thoroughly described in Example 14.

FIG. 13 shows the effect on the anticoagulant activity of 400 nMbetrixaban by varying concentrations of the antidote, des-Glaanhydro-fXa, in an aPTT assay using a standard coagulation timer. Theassay protocol is described in Example 3. The data shows that theantidote of fXa inhibitor substantially reverses the inhibition of fXaby 400 nM of betrixaban. The EC₅₀ of the antidote was estimated to beabout 656 nM with 400 nM betrixaban.

FIG. 14 shows the map of the DNA construct for expression of the fXatriple mutant (SEQ ID NO. 12) in CHO cells. Plasmid DNA was linearizedand transfected into CHO dhfr(−) cells. Cells were selected usingtetrahydrofolate (HT) deficient media plus methotrexate (MTX). Stableclones were screened for high protein expression by ELISA. The fXatriple mutant was produced in serum free medium and purified bycombination of ion exchange and affinity columns. The numbering in themap was based on polynucleotide sequence encoding human fX SEQ ID NO. 1.For example, an alanine mutation at the active site S419 (SEQ ID NO. 1)is equivalent to the mutation at S379 (SEQ ID NO. 3) of mature human fXdiscussed throughout the application and more particularly, Example 7.Primers used to construct the polynucleotide encoding the r-Antidotetriple mutant are listed in Table 28.

FIG. 15 shows a Western blot of purified r-Antidote by ion exchange andaffinity purification. Upon reduction of the disulfide bond whichconnects the light and heavy chains, the r-Antidote heavy chain migratesat expected molecular weight similar to that of plasma derived fXa.Deletion of 6-39 aa in the Gla-domain of fXa mutant results in a lowermolecular weight band of the r-Antidote light chain compared to normalFXa.

FIG. 16 shows betrixaban plasma level in mice (n=7-10 per group) afteroral administration of betrixaban alone (15 mg/kg), or betrixaban (15mg/kg) followed by intravenous injection (300 μg, IV) of plasma derivedantidote (pd-Antidote) prepared according to Example 1. pd-Antidote wasadministered 5 minutes prior to the 1.5 hr. time point, and mouse bloodsamples (0.5 mL) were taken at 1.5, 2.0, and 4.0 hrs following oraladministration of betrixaban. Whole blood INR, betrixaban and antidoteplasma levels were analyzed. Betrixaban level (Mean±SEM) in mouse plasmawas plotted as a function of time for mice after 15 mg/kg (open square)and 15 mg/kg followed by antidote injection (open circle). The PK-PDcorrelation of antidote treated group at 1.5 hr time point (5 min afterantidote injection) was summarized in Table A. Single injection of theantidote resulted in >50% reduction of functional betrixaban based onINR measurements. This is more thoroughly described in Example 8.

FIGS. 17A and B shows the results of a mouse experiment with purifiedr-Antidote (n=4-10 per group). Betrixaban level in mouse plasma (FIG.17A) and whole blood INR (FIG. 17B) were compared after oraladministration of betrixaban alone (15 mg/kg) or betrixaban (15 mg/kg)followed by intravenous injection (300 μg) of r-antidote. Mean valuesfor each treated group were indicated. As summarized in Table B, singleIV injection of the r-antidote resulted in >50% correction of ex vivowhole blood INR, justifying effective neutralization of fXa inhibitorsby the antidote via a single or multiple injections or other regimes.These results demonstrate that the fXa variants of this invention havepotential of acting as universal antidotes to reverse the anticoagulanteffect of fXa inhibitors in patients with bleeding or other medicalemergencies. This is more thoroughly described in Example 8.

FIG. 18 shows r-Antidote reversal of the inhibitory effect of enoxaparinin a 96-well turbidity change clotting assay. The results areessentially similar to pd-Antidote (FIG. 11) indicating both fXaderivatives have comparable functional antidote activity. 50 nMr-Antidote substantially corrected (>75%) the inhibitory effect of 1.25U/mL enoxaparin. The assay protocol is presented in Example 11.

FIG. 19 shows r-Antidote reversal of the inhibitory effect of lowmolecular weight heparin (LMWH) as tested in human plasma clottingassay. Both FIGS. 18 and 19 are discussed in Example 11.

FIG. 20 shows the r-Antidote reversal of the anticoagulation effect ofrivaroxaban. This is more thoroughly discussed in Example 12.

FIG. 21 shows the alignment of the polynucleotide sequence (SEQ ID NO:16) and translated polypeptide sequence (SEQ ID NO: 43) of r-Antidote.

FIGS. 22A and B shows the results of a mouse experiment with a single IVinjection (1 injection) or two injections (2 injections) of ther-antidote (n=5 per group, 312 ug/200 ul r-Antidote). Betrixaban levelin plasma (FIG. 22A) were compared after oral administration ofbetrixaban (15 mg/kg) followed by intravenous injection of vehicle orr-Antidote (see Example 8 for details). As shown in FIG. 22A, a singleIV injection of r-Antidote increased betrixaban level in plasma by morethan 8 fold compared to vehicle control (control_(—)1), indicating theability of the antidote to effectively bind betrixaban in vivo. A secondinjection of the antidote further increased betrixaban level by lessthan 2 fold compared to the single injection, indicating limiting amountof betrixaban in mouse blood and reversal of its anticoagulant effect bythe antidote. FIG. 22B demonstrates that measured INR decreases as theratio of antidote/betrixaban increases in mouse plasma following singleand double injections of the antidote.

FIG. 23 shows the plasma concentration-time profile of the r-Antidote inSprague-Dawley rats based on the data collected in Example 15.

FIG. 24 shows the plasma concentration-time profile of the r-Antidote inrhesus monkeys based on the data collected in Example 15.

DETAILED DESCRIPTION OF THE INVENTION I. Definitions

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of tissue culture, immunology,molecular biology, microbiology, cell biology and recombinant DNA, whichare within the skill of the art. See, e.g., Sambrook and Russell eds.(2001) Molecular Cloning: A Laboratory Manual, 3^(rd) edition; theseries Ausubel et al. eds. (2007) Current Protocols in MolecularBiology; the series Methods in Enzymology (Academic Press, Inc., N.Y.);MacPherson et al. (1991) PCR 1: A Practical Approach (IRL Press atOxford University Press); MacPherson et al. (1995) PCR 2: A PracticalApproach; Harlow and Lane eds. (1999) Antibodies, A Laboratory Manual;Freshney (2005) Culture of Animal Cells: A Manual of Basic Technique,5^(th) edition; Gait ed. (1984) Oligonucleotide Synthesis; U.S. Pat. No.4,683,195; Hames and Higgins eds. (1984) Nucleic Acid Hybridization;Anderson (1999) Nucleic Acid Hybridization; Hames and Higgins eds.(1984) Transcription and Translation; Immobilized Cells and Enzymes (IRLPress (1986)); Perbal (1984) A Practical Guide to Molecular Cloning;Miller and Calos eds. (1987) Gene Transfer Vectors for Mammalian Cells(Cold Spring Harbor Laboratory); Makrides ed. (2003) Gene Transfer andExpression in Mammalian Cells; Mayer and Walker eds. (1987)Immunochemical Methods in Cell and Molecular Biology (Academic Press,London); Herzenberg et al. eds (1996) Weir's Handbook of ExperimentalImmunology; Manipulating the Mouse Embryo: A Laboratory Manual, 3^(rd)edition (Cold Spring Harbor Laboratory Press (2002)).

All numerical designations, e.g., pH, temperature, time, concentration,and molecular weight, including ranges, are approximations which arevaried (+) or (−) by increments of 0.1. It is to be understood, althoughnot always explicitly stated that all numerical designations arepreceded by the term “about”. It also is to be understood, although notalways explicitly stated, that the reagents described herein are merelyexemplary and that equivalents of such are known in the art.

As used in the specification and claims, the singular form “a”, “an” and“the” include plural references unless the context clearly dictatesotherwise. For example, the term “a pharmaceutically acceptable carrier”includes a plurality of pharmaceutically acceptable carriers, includingmixtures thereof.

As used herein, the term “comprising” is intended to mean that thecompositions and methods include the recited elements, but do notexclude others. “Consisting essentially of” when used to definecompositions and methods, shall mean excluding other elements of anyessential significance to the combination for the intended use. Thus, acomposition consisting essentially of the elements as defined hereinwould not exclude trace contaminants from the isolation and purificationmethod and pharmaceutically acceptable carriers, such as phosphatebuffered saline, preservatives, and the like. “Consisting of” shall meanexcluding more than trace elements of other ingredients and substantialmethod steps for administering the compositions of this invention.Embodiments defined by each of these transition terms are within thescope of this invention.

A “subject” of diagnosis or treatment is a cell or a mammal, including ahuman. Non-human animals subject to diagnosis or treatment include, forexample, murine, such as rats, mice, canine, such as dogs, leporids,such as rabbits, livestock, sport animals, and pets.

The term “protein” and “polypeptide” are used interchangeably and intheir broadest sense to refer to a compound of two or more subunit aminoacids, amino acid analogs or peptidomimetics. The subunits may be linkedby peptide bonds. In another embodiment, the subunit may be linked byother bonds, e.g., ester, ether, etc. A protein or peptide must containat least two amino acids and no limitation is placed on the maximumnumber of amino acids which may comprise a protein's or peptide'ssequence. As used herein the term “amino acid” refers to either naturaland/or unnatural or synthetic amino acids, including glycine and boththe D and L optical isomers, amino acid analogs and peptidomimetics.Single letter and three letter abbreviations of the naturally occurringamino acids are listed below. A peptide of three or more amino acids iscommonly called an oligopeptide if the peptide chain is short. If thepeptide chain is long, the peptide is commonly called a polypeptide or aprotein.

1-Letter 3-Letter Amino Acid Y Tyr L-tyrosine G Gly L-glycine F PheL-phenylalanine M Met L-methionine A Ala L-alanine S Ser L-serine I IleL-isoleucine L Leu L-leucine T Thr L-threonine V Val L-valine P ProL-proline K Lys L-lysine H His L-histidine Q Gln L-glutamine E GluL-glutamic acid W Trp L-tryptohan R Arg L-arginine D Asp L-aspartic acidN Asn L-asparagine C Cys L-cysteine

“Factor Xa” or “fXa” or “fXa protein” refers to a serine protease in theblood coagulation pathway, which is produced from the inactive factor X(fX). Factor Xa is activated by either factor IXa with its cofactor,factor VIIIa, in a complex known as intrinsic Xase, or factor VIIa withits cofactor, tissue factor, in a complex known as extrinsic Xase. fXaforms a membrane-bound prothrombinase complex with factor Va and is theactive component in the prothrombinase complex that catalyzes theconversion of prothrombin to thrombin. Thrombin is the enzyme thatcatalyzes the conversion of fibrinogen to fibrin, which ultimately leadsto blood clot formation. Thus, the biological activity of fXa issometimes referred to as “procoagulant activity” herein.

Factor Xa is a two chain molecule linked by one disulfide bond betweenthe two chains. The heavy chain contains the serine protease,trypsin-like active site and the N-terminal activation peptide which isglycosylated. The heavy chain has at least three forms, α, β, and γ,which differ due to the cleavage of a C-terminal peptide in the heavychain (Aronson, D. L. et al. Proc. Soc. Exp. Biol. Med. 137(4):1262-1266(1971); Mertens, K. Et al., Biochem. J. 185:647-658 (1980)). This isfurther discussed in sections describing fX and fXa derivatives.

The nucleotide sequence coding human factor X (“fX”) can be found inGenBank, with accession number “NM_(—)000504”, and is listed in FIG. 1 band SEQ ID No. 2. The corresponding amino acid sequence and domainstructure of fX are described in Leytus et al, Biochemistry, 1986,25:5098-5102. The domain structure of mature IX is also described inVenkateswarlu, D. et al, Biophysical Journal, 2002, 82:1190-1206. Uponcatalytic cleavage of the first 52 residues (amino acids 143 to 194 ofSEQ ID NO. 3) of the heavy chain, fX is activated to fXa (SEQ ID NO. 6).FXa contains a light chain (SEQ ID NO. 8) and a heavy chain (SEQ ID NO.9). The first 45 amino acid residues (residues 1-45 of SEQ ID NO. 6) ofthe light chain is called the Gla domain because it contains 11post-translationally modified γ-carboxyglutamic acid residues (Gla). Italso contains a short (6 amino acid residues) aromatic stack sequence(residues 40-45 of SEQ ID NO. 6). Chymotrypsin digestion selectivelyremoves the 1-44 residues resulting Gla-domainless fXa (SEQ ID NO. 4).The serine protease catalytic domain of fXa locates at the C-terminalheavy chain. The heavy chain of fXa is highly homologous to other serineproteases such as thrombin, trypsin, and activated protein C.

The domain structure of mature factor X may be found in Venkateswarlu D.et al, Biophysical J., 2002, 82, 1190-1206, which is hereby incorporatedby reference in its entirety. The amino acid numbering in this figure isthe same as in FIG. 3. The tripeptide of Arg140-Lys141-Arg142 (SEQ IDNO: 40) (the RKR triplet (SEQ ID NO: 40) as shown in FIG. 1) thatconnects the light chain to the activation peptide is not shown becausethe form that lacks the tripeptide is predominant incirculation bloodplasma. Individual domains are shown in boxes. This includes amino acids1-45 in FIG. 2 (SEQ ID NO. 3). Functionally important catalytic residuesare circled, and “g” represents Gla (g-carboxyglutamic acid) residue.

“Native fXa” or “wild-type fXa” refers to the fXa naturally present inplasma or being isolated in its original, unmodified form, whichprocesses the biological activity of activating prothrombin thereforepromoting formation of blood clot. The term includes naturally occurringpolypeptides isolated from tissue samples as well as recombinantlyproduced fXa. “Active fXa” refers to fXa having the biological activityof activating prothrombin. “Active fXa” may be a native fXa or modifiedfXa that retains procoagulant activity.

“fX Derivatives” or “modified fXa” derivatives refers to fX proteinsthat have been modified such that they bind, either directly orindirectly, to a factor Xa inhibitor and do not assemble into theprothrombinase complex. Examples include, but are not limited to, thepolypeptide in SEQ ID NO. 19. Included within the definition of fXderivatives are fXa derivatives discussed below. In some embodiments,the modified derivative may also use the term “Factor Xai”.

“fXa Derivatives” or “modified fXa” or “derivatives of a factor Xaprotein” refers to fXa proteins that have been modified such that theybind, either directly or indirectly, to a factor. Xa inhibitor and donot assemble into the prothrombinase complex. Structurally, thederivatives are modified to provide either no procoagulant activity orreduced procoagulant activity. “Procoagulant activity” is referred toherein as an agent's ability to cause blood coagulation or clotformation. Reduced procoagulant activity means that the procoagulantactivity has been reduced by at least about 50%, or more than about 90%,or more than about 95% as compared to wild-type fXa during the sameperiod. For example, recombinant fX-S395A essentially has noprocoagulant activity as measured by in vitro assays, such as fXaactivity assays.

The derivatives have modified active sites and a modified Gla domain.Additional modifications are also contemplated. It is contemplated thatsuch modifications may be made in one or more of the following ways:deletion of one or more of the amino acid from the sequence,substitution of one or more amino acid residues with one or moredifferent amino acid residues, and/or manipulation of one or more aminoacid side chains or its “C” or “N” terminals.

The term “active site” refers to the part of an enzyme or antibody wherea chemical reaction occurs. A “modified active site” is an active sitethat has been modified structurally to provide the active site withincreased or decreased chemical reactivity or specificity. It iscontemplated that the active site includes not only the actual site butalso a domain containing the active site. Examples of active sitesinclude, but are not limited to, the catalytic domain of human factor Xcomprising the 235-488 amino acid residues (FIG. 1), and the catalyticdomain of human factor Xa comprising the 195-488 amino acid residues(FIGS. 2 and 3). The catalytic triad equivalent to H57-D102-S195 inchymotrypsin numbering are located at His236, Asp282, and Ser379.Examples of modified active site include, but are not limited to, thecatalytic triad residues individually or in combination. Onemodification relates to a fXa derivative having a modified active siteSer379/A. Additional examples, include modifications to the catalyticdomain of human factor Xa comprising 195-448 amino acid residues in SEQID NOS. 10, 11, 12, 13, or 15 with at least one amino acid substitutionat position Arg306, Glu310, Arg347, Lys351, Lys414, or Arg424.

As stated above, the derivatives of the invention may have modified Gladomains or have the entire Gla domain removed. Examples of fXaderivatives suitable as antidotes in the methods of this invention areGla-domainless fX (SEQ ID NO. 19), Gla-domainless fXa (SEQ ID NOS. 4, 5,20, 21, 22, 23, 24, or 25), Gla-deficient fXa (SEQ ID NO. 7 withmodifications described herein), fXa with modifications at the catalyticsite (SEQ ID NOS. 10 or 11), and fXa with modifications at the sitesknown to be important for fV/fVa interaction or fVIII/fVIIIa interaction(SEQ ID NOS. 4, 5, 7, 10, or 11 with at least one amino acidsubstitution at position Arg306, Glu310, Arg347, Lys351, Lys414 orArg424), as described in detail herein. Further examples of the fX andfXa derivatives contemplated by this invention are provided below.

“Gla-domainless” or “des-Gla” refers to fXa or fX that does not have aGla-domain and encompasses fXa and fX derivatives bearing othermodification(s) in addition to the removal of the Gla-domain. Examplesof Gla-domainless fXa in this invention include, but are not limited to,fXa derivative lacking the 1-39 amino acid residues of SEQ ID NO. 3; fXaderivative lacking the 6-39 amino acid residues of SEQ ID NO. 3,corresponding to a fXa mutant expressed in CHO cells described in moredetails below (SEQ ID NO. 12, Table 12); fXa derivative lacking the 1-44amino acid residues of SEQ ID NO. 3, corresponding to des-Gla fXa afterchymotryptic digestion of human fXa (SEQ ID NO. 4, FIG. 3); and fXaderivative lacking the entire 1-45 Gla-domain residues of SEQ ID NO. 3as described in Padmanabhan et al, Journal Mol. Biol., 1993, 232:947-966(SEQ ID NO 5). Other examples include des-Gla anhydro fXa (SEQ ID NO.10, Table 10) and des-Gla fXa-S379A (SEQ ID NO. 11, Table 11).

In some embodiments, the des-Gla fXa comprises at least amino acidresidues 40 to 448 of SEQ ID NO. 3 or an equivalent thereof. In someembodiment, the des-Gla fXa comprises at least amino acid residues 45 to488 (SEQ ID NO. 4) or 46 to 488 (SEQ ID NO. 5) of SEQ ID NO. 3 orequivalents thereof.

In some embodiment, the des-Gla fXa comprises at least amino acidresidues 40 to 139 and 195 to 448 of SEQ ID NO. 3 or equivalentsthereof. In some embodiment, the des-Gla fXa comprises at least aminoacid residues 45 to 139 and 195 to 448 of SEQ ID NO. 3 or equivalentsthereof. In another embodiment, the des-Gla fXa comprises at least aminoacid residues 46 to 139 and 195 to 448 of SEQ ID NO. 3 or equivalentsthereof.

“Gla-deficient” refers to fXa or fX with reduced number of free sidechain γ-carboxyl groups in its Gla-domain. Like Gla-domainless fXa,Gla-deficient fXa can also bear other modifications. Gla-deficient fXaincludes uncarboxylated, undercarboxylated and decarboxylated fXa.“Uncarboxylated fXa” or “decarboxylated fXa” refers to fXa derivativesthat do not have the γ-carboxy groups of the γ-carboxyglutamic acidresidues of the Gla domain, such as fXa having all of its Gla domainγ-carboxyglutamic acid replaced by different amino acids, or fXa havingall of its side chain γ-carboxyl removed or masked by means such asamination, esterification, etc. For recombinantly expressed protein,uncarboxylated fXa is, sometimes, also called non-carboxylated fXa.“Undercarboxylated fXa” refers to fXa derivatives having reduced numberof γ-carboxy groups in the Gla domain as compared with wild-type fXa,such as fXa having one or more but not all of its Gla domainγ-carboxyglutamic acids replaced by one or more different amino acids,or fXa having at least one but not all of its side chain γ-carboxylremoved or masked by means such as amination and esterification, etc.

The domain structure of human Gla-domainless factor Xa may be found inPadmanabhan et al., J. Mol. Biol., 1993, 232, 947-966, which is herebyincorporated by reference in its entirety. The numbering of the aminoacid is based on topological equivalences with chymotrypsin, where, forexample, Ser195 corresponds to Ser379 in FIG. 2 when the human mature fXnumbering is used. Insertions are indicated with letters, and deletionsare indicated by 2 successive numberings. 300 are added to light chainnumbering to differentiate from the heavy chain numbering. β363 isβ-hydroxy aspartate. Slashes indicate proteolytic cleavages observed incrystallized material. The sequence of Gla-domainless fXa lacking the1-45 amino acid residues based mature fX (SEQ ID NO. 3) is listed in SEQID NO. 5.

In one embodiment of the invention, all or part of the “activationpeptide” (AP) is removed. What is meant by activation peptide is aminoacids 183 to 234 of SEQ ID NO. 1 or alternatively, amino acids 143-194of SEQ ID NO. 3.

In another embodiment, a signal peptide is included in the derivative.The term “signal peptide” refers to a factor X signal peptide (SEQ IDNO. 18), a transferrin signal peptide (amino acid residues SP1 to SP19of SEQ ID NO. 27), or a prothrombin signal peptide (amino acid residuesSP1 to SP24 of SEQ ID NO. 26).

In one embodiment, the fXa derivative may lack a light chain of fXa butstill contains a serine protease catalytic domain present in the heavychain. In addition chimeras with other serine protease catalytic domainmay be used to make substitutions in the heavy chain.

A “β-peptide” refers to all or a portion of the heavy chain.

“pd-Antidote” or “plasma-derived antidote” refers to the des-Gla anhydrofXa derivative and has the amino acid residues of SEQ ID NO. 10.

“r-Antidote” or “recombinant antidote” refers to a fXa derivativelacking the 6-39 amino acid residues of SEQ ID NO. 3, corresponding to afXa mutant expressed in CHO cells and after removal of the linkerdescribed in more detail below (SEQ ID NO. 13, Table 13).

“Anticoagulant agents” or “anticoagulants” are agents that inhibit bloodclot formation. Examples of anticoagulant agents include, but are notlimited to, specific inhibitors of thrombin, factor IXa, factor Xa,factor XIa, factor XIIa or factor VIIa, heparin and derivatives, vitaminK antagonists, and anti-tissue factor antibodies. Examples of specificinhibitors of thrombin include hirudin, bivalirudin (Angiomax®),argatroban and lepirudin (Refludan®). Examples of heparin andderivatives include unfractionated heparin (UFH), low molecular weightheparin (LMWH), such as enoxaparin (Lovenox®), dalteparin (Fragmin®),and danaparoid (Orgaran®); and synthetic pentasaccharide, such asfondaparinux (Arixtra®). Examples of vitamin K antagonists includewarfarin (Coumadin®), phenocoumarol, acenocoumarol (Sintrom®),clorindione, dicumarol, diphenadione, ethyl biscoumacetate,phenprocoumon, phenindione, and tioclomarol. In one embodiment, theanticoagulant is an inhibitor of factor Xa. In one embodiment, theanticoagulant is betrixaban.

“Anticoagulant therapy” refers to a therapeutic regime that isadministered to a patient to prevent undesired blood clots orthrombosis. An anticoagulant therapy comprises administering one or acombination of two or more anticoagulant agents or other agents at adosage and schedule suitable for treating or preventing the undesiredblood clots or thrombosis in the patient.

The term “factor Xa inhibitors” or “inhibitors of factor Xa” refer tocompounds that can inhibit, either directly or indirectly, thecoagulation factor Xa's activity of catalyzing conversion of prothrombinto thrombin in vitro and/or in vivo. Examples of known fXa inhibitorsinclude, without limitation, edoxaban, fondaparinux, idraparinux,biotinylated idraparinux, enoxaparin, fragmin, NAP-5, rNAPc2, tissuefactor pathway inhibitor, DX-9065a (as described in, e.g., Herbert, J.M., et al, J Pharmacol Exp Ther. 1996 276(3):1030-8), YM-60828 (asdescribed in, e.g., Taniuchi, Y., et al, Thromb Haemost. 199879(3):543-8), YM-150 (as described in, e.g., Eriksson, B. I. et. al,Blood 2005; 106(11), Abstract 1865), apixaban, rivaroxaban, PD-348292(as described in, e.g., Pipeline Insight: Antithrombotics—Reaching theUntreated Prophylaxis Market, 2007), otamixaban, razaxaban (DPC906), BAY59-7939 (as described in, e.g., Turpie, A. G., et al, J. Thromb.Haemost. 2005, 3(11):2479-86), edoxaban (as described in, e.g., Hylek EM, Curr Opin Invest Drugs 2007 8(9):778-783), LY517717 (as described in,e.g., Agnelli, G., et al, J. Thromb. Haemost. 2007 5(4):746-53),GSK913893, betrixaban (as described below) and derivatives thereof. Lowmolecular weight heparin (“LMWH”) is also considered a factor Xainhibitor.

In one embodiment, the factor Xa inhibitor is selected from betrixaban,rivaroxaban, apixaban, edoxaban, LMWH, and combinations thereof.

The term “betrixaban” refers to the compound“[2-({4-[(dimethylamino)iminomethyl]phenyl}carbonylamino)-5-methoxyphenyl]-N-(5-chloro(2-pyridyl)carboxamide”or pharmaceutically acceptable salts thereof.“[2-({4-[(dimethylamino)iminomethyl]phenyl}carbonylamino)-5-methoxyphenyl]-N-(5-chloro(2-pyridyl))carboxamide”refers to the compound having the following structure:

or a tautomer or pharmaceutically acceptable salt thereof.

Betrixaban is described in U.S. Pat. Nos. 6,376,515 and 6,835,739 andU.S. Patent Application Publication No. 2007/0112039, filed on Nov. 7,2006, the contents of which are incorporated herein by reference.Betrixaban is known to be a specific inhibitor of factor Xa.

As used herein, the term “antidote” or “antidote to a factor Xainhibitor” refers to molecules, such as derivatives of fX or fXa, whichcan substantially neutralize or reverse the coagulation inhibitoryactivity of a fXa inhibitor by competing with active fXa to bind withavailable fXa inhibitors. Examples of the antidotes of this inventionare fXa derivatives with reduced phospholipid membrane binding, such asdes-Gla fXa or Gla-deficient fXa, and fXa derivatives with reducedcatalytic activity, such as the active site modified fXa derivatives,and derivatives with reduced interaction with fV/Va, or fVIII/fVIIIa.Examples of antidotes of the invention with reduced membrane binding andreduced catalytic activity include, but are not limited to, des-Glaanhydro-fXa by chymotryptic digestion of anhydro-fXa (as described inExample 1); des-Gla fXa-S379A (S195A in chymotrypsin numbering) bymutagenesis (as described in Example 6).

Other examples of antidotes of the invention include proteins orpolypeptides containing serine protease catalytic domains which possesssufficient structural similarity to fXa catalytic domain and aretherefore capable of binding small molecule fXa inhibitors. Examplesinclude, but are not limited to, thrombin which binds to the fXainhibitor GSK913893 (Young R., et al., Bioorg. Med. Chem. Lett. 2007,17(10): 2927-2930); plasma kallikrein which binds to the fXa inhibitorapixaban (Luettgen J., et al., Blood, 2006, 108(11) abstract 4130); andtrypsin (or its bacterial homolog subtilisin) which binds the fXainhibitor C921-78 with subnanomolar affinity (Kd=500 pM) (Betz A, et al,Biochem., 1999, 38(44):14582-14591).

In one embodiment, the derivative of the invention binds, eitherdirectly or indirectly to a factor Xa inhibitor. The terms “binding,”“binds,” “recognition,” or “recognize” as used herein are meant toinclude interactions between molecules that may be detected using, forexample, a hybridization assay. The terms are also meant to include“binding” interactions between molecules. Interactions may be, forexample, protein-protein, protein-nucleic acid, protein-small moleculeor small molecule-nucleic acid in nature. Binding may be “direct” or“indirect”. “Direct” binding comprises direct physical contact betweenmolecules. “Indirect” binding between molecules comprises the moleculeshaving direct physical contact with one or more intermediate moleculessimultaneously. For example, it is contemplated that derivatives of theinvention indirectly bind and substantially neutralize low molecularweight heparin and other indirect inhibitors of factor Xa. This bindingcan result in the formation of a “complex” comprising the interactingmolecules. A “complex” refers to the binding of two or more moleculesheld together by covalent or non-covalent bonds, interactions or forces.

“Neutralize,” “reverse” or “counteract” the activity of an inhibitor offXa or similar phrases refer to inhibit or block the factor Xainhibitory or anticoagulant function of a fXa inhibitor. Such phrasesrefer to partial inhibition or blocking of the function, as well as toinhibiting or blocking most or all of fXa inhibitor activity, in vitroand/or in vivo. These terms also refer to corrections of at least about20% of fXa inhibitor dependent pharmacodynamic or surrogate markers.Examples of markers include, but are not limited to INR, PR, aPTT, ACT,anti-fXa units, thrombin generation (Technothrombin TGA),thromboelastogrpahy, CAT (calibrated automated thrombogram) and thelike.

In certain embodiments, the factor Xa inhibitor is neutralizedsubstantially (or corrected as just described) meaning that its abilityto inhibit factor Xa, either directly or indirectly, is reduced by atleast about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%.

The term “phospholipid membrane binding” refers to an active fXa'sability to bind to the negatively charged phospholipid membrane or othercellular membrane, such as platelets, in the presence of Ca²⁺ ions. Thisbinding is mediated by the γ-carboxyglutamic acid residues in the Gladomain of fXa.

The term “reduced interaction” refers to fXa derivative's diminishedability to bind or form a complex with ions or other co-factors whichnormally binds or complexes with wild fXa. Examples of such interactioninclude but are not limited to fXa's binding with Ca²⁺ ions andphospholipid membrane, interaction with fV/fVa, or fVIII/fVIIIa, etc. Itis preferred that the interaction of a fXa derivative with the ions orother co-factors is reduced to 50% of that of a wild fXa. Morepreferably, the interaction is reduced to 10%, 1%, and 0.1% of that of awild-type fXa. This refers to the derivatives' ability to “assemble intothe prothrombinase complex.”

“fXa inhibitor binding activity” refers to a molecule's ability to bindan inhibitor of fXa. An antidote of the present invention possesses fXainhibitor binding activity, whether it is directly or indirectly.

The term “circulating half life” or “plasma half life” refers to thetime required for the plasma concentration of an antidote thatcirculates in the plasma to reduce to half of its initial concentrationafter a single administration or following cessation of infusion.

The term “conjugated moiety” refers to a moiety that can be added to afXa derivative by forming a covalent bond with a residue of the fXaderivative. The moiety may bond directly to a residue of the fXaderivative or may form a covalent bond with a linker which in turn formsa covalent bond with a residue of the fXa derivative.

As used herein, an “antibody” includes whole antibodies and any antigenbinding fragment or a single chain thereof. Thus the term “antibody”includes any protein or peptide containing molecule that comprises atleast a portion of an immunoglobulin molecule. Examples of such include,but are not limited to a complementarity determining region (CDR) of aheavy or light chain or a ligand binding portion thereof, a heavychainor light chain variable region, a heavy chain or light chain constantregion, a framework (FR) region, or any portion thereof, or at least oneportion of a binding protein.

The antibodies can be polyclonal or monoclonal and can be isolated fromany suitable biological source, e.g., murine, rat, sheep and canine.

A “composition” is intended to mean a combination of active agent andanother compound or composition, inert (for example, a detectable agentor label) or active, such as an adjuvant.

A “pharmaceutical composition” is intended to include the combination ofan active agent with a carrier, inert or active, making the compositionsuitable for diagnostic or therapeutic use in vitro, in vivo or ex vivo.

“An effective amount” refers to the amount of derivative sufficient toinduce a desired biological and/or therapeutic result. That result canbe alleviation of the signs, symptoms, or causes of a disease, or anyother desired alteration of a biological system. In the presentinvention, the result will typically involve one or more of thefollowing: neutralization of a fXa inhibitor that has been administeredto a patient, reversal of the anticoagulant activity of the fXainhibitor, removal of the fXa inhibitor from the plasma, restoration ofhemostasis, and reduction or cessation of bleeding. The effective amountwill vary depending upon the specific antidote agent used, the specificfXa inhibitor the subject has been administered, the dosing regimen ofthe fXa inhibitor, timing of administration of the antidote, the subjectand disease condition being treated, the weight and age of the subject,the severity of the disease condition, the manner of administration andthe like, all of which can be determined readily by one of ordinaryskill in the art.

Effective unit doses of the antidotes described herein are described inU.S. Provisional Application Ser. No. 61/225,887, filed on Jul. 15, 2009and entitled “Unit Dose Formulation of Antidotes for Factor XaInhibitors and Methods of Using the Same,” which is hereby incorporatedby reference in its entirety. Specifically, it is contemplated that theunit dose formulation comprising a pharmaceutically acceptable carrierand a two chain polypeptide comprising the amino acid sequence of SEQ IDNO. 13 or a polypeptide having at least 80% homology to SEQ ID NO. 13 isan amount from about 10 milligrams to about 2 grams or from about 100milligrams to about 1.5 grams or from about 200 milligrams to about 1gram or from about 400 milligrams to about 900 milligrams. It is furthercontemplated that other antidotes of the invention will be dosed in asimilar manner.

As used herein, the terms “treating,” “treatment” and the like are usedherein to mean obtaining a desired pharmacologic and/or physiologiceffect. The effect may be prophylactic in terms of completely orpartially preventing a disorder or sign or symptom thereof, and/or maybe therapeutic in terms of a partial or complete cure for a disorderand/or adverse effect attributable to the disorder.

“Treating” also covers any treatment of a disorder in a mammal, andincludes: (a) preventing a disorder from occurring in a subject that maybe predisposed to a disorder, but may have not yet been diagnosed ashaving it, e.g., prevent bleeding in a patient with anticoagulantoverdose; (b) inhibiting a disorder, i.e., arresting its development,e.g., inhibiting bleeding; or (c) relieving or ameliorating thedisorder, e.g., reducing bleeding.

As used herein, to “treat” further includes systemic amelioration of thesymptoms associated with the pathology and/or a delay in onset ofsymptoms. Clinical and sub-clinical evidence of “treatment” will varywith the pathology, the individual and the treatment. Further, the term“prevent” also refers to inhibiting.

“Administration” can be effected in one dose, continuously orintermittently throughout the course of treatment. Methods ofdetermining the most effective means and dosage of administration areknown to those of skill in the art and will vary with the compositionused for therapy, the purpose of the therapy, the target cell beingtreated, and the subject being treated. Single or multipleadministrations can be carried out with the dose level and pattern beingselected by the treating physician. Suitable dosage formulations andmethods of administering the agents are known in the art.

The agents and compositions of the present invention can be used in themanufacture of medicaments and for the treatment of humans and otheranimals by administration in accordance with conventional procedures,such as an active ingredient in pharmaceutical compositions.

An agent of the present invention can be administered for therapy by anysuitable route, specifically by parental (including subcutaneous,intramuscular, intravenous and intradermal) administration. It will alsobe appreciated that the preferred route will vary with the condition andage of the recipient, and the disease being treated.

One can determine if the method, i.e., inhibition or reversal of afactor Xa inhibitor is achieved, by a number of in vitro assays, such asthrombin generation assay, and anti-fXa units, and clinical clottingassays such as aPTT, PT and ACT.

The term “isolated” as used herein with respect to nucleic acids, suchas DNA or RNA, refers to molecules separated from other DNAs or RNAs,respectively that are present in the natural source of themacromolecule. The term “isolated nucleic acid” is meant to includenucleic acid fragments which are not naturally occurring as fragmentsand would not be found in the natural state. The term “isolated” is alsoused herein to refer to polypeptides and proteins that are isolated fromother cellular proteins and is meant to encompass both purified andrecombinant polypeptides. In other embodiments, the term “isolated”means separated from constituents, cellular and otherwise, in which thecell, tissue, polynucleotide, peptide, polypeptide, protein, antibody orfragment(s) thereof, which are normally associated in nature. Forexample, an isolated cell is a cell that is separated from tissue orcells of dissimilar phenotype or genotype. As is apparent to those ofskill in the art, a non-naturally occurring polynucleotide, peptide,polypeptide, protein, antibody or fragment(s) thereof, does not require“isolation” to distinguish it from its naturally occurring counterpart.

As used herein, the term “equivalent thereof” when referring to areference protein, polypeptide or nucleic acid, intends those havingminimal homology while still maintaining desired functionality. It iscontemplated that any modified protein mentioned herein also includesequivalents thereof. For example, the homology can be, at least 75%homology and alternatively, at least 80%, or alternatively at least 85%,or alternatively at least 90%, or alternatively at least 95%, oralternatively 98% percent homology and exhibit substantially equivalentbiological activity to the reference polypeptide or protein. Apolynucleotide or polynucleotide region (or a polypeptide or polypeptideregion) has a certain percentage (for example, 80%, 85%, 90%, or 95%) of“sequence identity” to another sequence means that, when aligned, thatpercentage of bases (or amino acids) are the same in comparing the twosequences. It should be noted that when only the heavy chain of fXa (ora related serine protease) is used, the overall homology might be lowerthan 75%, such as, for example, 65% or 50% however, the desiredfunctionality remains. This alignment and the percent homology orsequence identity can be determined using software programs known in theart, for example those described in CURRENT PROTOCOLS IN MOLECULARBIOLOGY (F. M. Ausubel et al., eds., 1987) Supplement 30, section7.7.18, Table 7.7.1. Preferably, default parameters are used foralignment. A preferred alignment program is BLAST, using defaultparameters. In particular, preferred programs are BLASTN and BLASTP,using the following default parameters: Genetic code=standard;filter=none; strand=both; cutoff=60; expect=10; Matrix=BLOSUM62;Descriptions=50 sequences; sort by=HIGH SCORE; Databases=non-redundant,GenBank+EMBL+DDBJ+PDB+GenBank CDStranslations+SwissProtein+SPupdate+PIR.

The terms “polynucleotide” and “oligonucleotide” are usedinterchangeably and refer to a polymeric form of nucleotides of anylength, either deoxyribonucleotides or ribonucleotides or analogsthereof. Polynucleotides can have any three-dimensional structure andmay perform any function, known or unknown. The following arenon-limiting examples of polynucleotides: a gene or gene fragment (forexample, a probe, primer, EST or SAGE tag), exons, introns, messengerRNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinantpolynucleotides, branched polynucleotides, plasmids, vectors, isolatedDNA of any sequence, isolated RNA of any sequence, nucleic acid probesand primers. A polynucleotide can comprise modified nucleotides, such asmethylated nucleotides and nucleotide analogs. If present, modificationsto the nucleotide structure can be imparted before or after assembly ofthe polynucleotide. The sequence of nucleotides can be interrupted bynon-nucleotide components. A polynucleotide can be further modifiedafter polymerization, such as by conjugation with a labeling component.The term also refers to both double- and single-stranded molecules.Unless otherwise specified or required, any embodiment of this inventionthat is a polynucleotide encompasses both the double-stranded form andeach of two complementary single-stranded forms known or predicted tomake up the double-stranded form.

A polynucleotide is composed of a specific sequence of four nucleotidebases: adenine (A); cytosine (C); guanine (G); thymine (T); and uracil(U) for thymine when the polynucleotide is RNA. Thus, the term“polynucleotide sequence” is the alphabetical representation of apolynucleotide molecule. This alphabetical representation can be inputinto databases in a computer having a central processing unit and usedfor bioinformatics applications such as functional genomics and homologysearching.

“Homology” or “identity” or “similarity” refers to sequence similaritybetween two peptides or between two nucleic acid molecules. Homology canbe determined by comparing a position in each sequence which may bealigned for purposes of comparison. When a position in the comparedsequence is occupied by the same base or amino acid, then the moleculesare homologous at that position. A degree of homology between sequencesis a function of the number of matching or homologous positions sharedby the sequences. An “unrelated” or “non-homologous” sequence sharesless than 40% identity, or alternatively less than 25% identity, withone of the sequences of the present invention.

A polynucleotide or polynucleotide region (or a polypeptide orpolypeptide region) has a certain percentage (for example, 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, 98% or 99%) of “sequence identity” toanother sequence means that, when aligned, that percentage of bases (oramino acids) are the same in comparing the two sequences. This alignmentand the percent homology or sequence identity can be determined usingsoftware programs known in the art, for example those described inAusubel et al. eds. (2007) Current Protocols in Molecular Biology.Preferably, default parameters are used for alignment. One alignmentprogram is BLAST, using default parameters. In particular, programs areBLASTN and BLASTP, using the following default parameters: Geneticcode=standard; filter=none; strand=both; cutoff=60; expect=10;Matrix=BLOSUM62; Descriptions=50 sequences; sort by=HIGH SCORE;Databases=non-redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDStranslations+SwissProtein+SPupdate+PIR. Biologically equivalentpolynucleotides are those having the specified percent homology andencoding a polypeptide having the same or similar biological activity.

The term “a homolog of a nucleic acid” refers to a nucleic acid having anucleotide sequence having a certain degree of homology with thenucleotide sequence of the nucleic acid or complement thereof. A homologof a double stranded nucleic acid is intended to include nucleic acidshaving a nucleotide sequence which has a certain degree of homology withor with the complement thereof. In one aspect, homologs of nucleic acidsare capable of hybridizing to the nucleic acid or complement thereof.

A “gene” refers to a polynucleotide containing at least one open readingframe (ORF) that is capable of encoding a particular polypeptide orprotein after being transcribed and translated. Any of thepolynucleotide or polypeptide sequences described herein may be used toidentify larger fragments or full-length coding sequences of the genewith which they are associated. Methods of isolating larger fragmentsequences are known to those of skill in the art.

The term “express” refers to the production of a gene product.

As used herein, “expression” refers to the process by whichpolynucleotides are transcribed into mRNA and/or the process by whichthe transcribed mRNA is subsequently being translated into peptides,polypeptides, or proteins. If the polynucleotide is derived from genomicDNA, expression may include splicing of the mRNA in an eukaryotic cell.

The term “encode” as it is applied to polynucleotides refers to apolynucleotide which is said to “encode” a polypeptide if, in its nativestate or when manipulated by methods well known to those skilled in theart, it can be transcribed and/or translated to produce the mRNA for thepolypeptide and/or a fragment thereof. The antisense strand is thecomplement of such a nucleic acid, and the encoding sequence can bededuced therefrom.

A “peptide conjugate” refers to the association by covalent ornon-covalent bonding of one or more polypeptides and another chemical orbiological compound. In a non-limiting example, the “conjugation” of apolypeptide with a chemical compound results in improved stability orefficacy of the polypeptide for its intended purpose. In one embodiment,a peptide is conjugated to a carrier, wherein the carrier is a liposome,a micelle, or a pharmaceutically acceptable polymer.

“Liposomes” are microscopic vesicles consisting of concentric lipidbilayers. Structurally, liposomes range in size and shape from longtubes to spheres, with dimensions from a few hundred Angstroms tofractions of a millimeter. Vesicle-forming lipids are selected toachieve a specified degree of fluidity or rigidity of the final complexproviding the lipid composition of the outer layer. These are neutral(cholesterol) or bipolar and include phospholipids, such asphosphatidylcholine (PC), phosphatidylethanolamine (PE),phosphatidylinositol (PI), and sphingomyelin (SM) and other types ofbipolar lipids including but not limited todioleoylphosphatidylethanolamine (DOPE), with a hydrocarbon chain lengthin the range of 14-22, and saturated or with one or more double C═Cbonds. Examples of lipids capable of producing a stable liposome, alone,or in combination with other lipid components are phospholipids, such ashydrogenated soy phosphatidylcholine (HSPC), lecithin,phosphatidylethanolamine, lysolecithin, lysophosphatidylethanol-amine,phosphatidylserine, phosphatidylinositol, sphingomyelin, cephalin,cardiolipin, phosphatidic acid, cerebrosides,distearoylphosphatidylethan-olamine (DSPE), dioleoylphosphatidylcholine(DOPC), dipalmitoylphosphatidylcholine (DPPC),palmitoyloleoylphosphatidylcholine (POPC),palmitoyloleoylphosphatidylethanolamine (POPE) anddioleoylphosphatidylethanolamine4-(N-maleimido-methyl)cyclohexane-1-carb-oxylate (DOPE-mal). Additionalnon-phosphorous containing lipids that can become incorporated intoliposomes include stearylamine, dodecylamine, hexadecylamine, isopropylmyristate, triethanolamine-lauryl sulfate, alkyl-aryl sulfate, acetylpalmitate, glycerol ricinoleate, hexadecyl stereate, amphoteric acrylicpolymers, polyethyloxylated fatty acid amides, and the cationic lipidsmentioned above (DDAB, DODAC, DMRIE, DMTAP, DOGS, DOTAP (DOTMA), DOSPA,DPTAP, DSTAP, DC-Chol). Negatively charged lipids include phosphatidicacid (PA), dipalmitoylphosphatidylglycerol (DPPG),dioleoylphosphatidylglycerol and (DOPG), dicetylphosphate that are ableto form vesicles. Typically, liposomes can be divided into threecategories based on their overall size and the nature of the lamellarstructure. The three classifications, as developed by the New YorkAcademy Sciences Meeting, “Liposomes and Their Use in Biology andMedicine,” December 1977, are multi-lamellar vesicles (MLVs), smalluni-lamellar vesicles (SUVs) and large uni-lamellar vesicles (LUVs).

A “micelle” is an aggregate of surfactant molecules dispersed in aliquid colloid. A typical micelle in aqueous solution forms an aggregatewith the hydrophilic “head” regions in contact with surrounding solvent,sequestering the hydrophobic tail regions in the micelle center. Thistype of micelle is known as a normal phase micelle (oil-in-watermicelle). Inverse micelles have the head groups at the center with thetails extending out (water-in-oil micelle). Micelles can be used toattach a polynucleotide, polypeptide, antibody or composition describedherein to facilitate efficient delivery to the target cell or tissue.

The phrase “pharmaceutically acceptable polymer” refers to the group ofcompounds which can be conjugated to one or more polypeptides describedhere. It is contemplated that the conjugation of a polymer to thepolypeptide is capable of extending the half-life of the polypeptide invivo and in vitro. Non-limiting examples include polyethylene glycols,polyvinylpyrrolidones, polyvinylalcohols, cellulose derivatives,polyacrylates, polymethacrylates, sugars, polyols and mixtures thereof.

A “gene delivery vehicle” is defined as any molecule that can carryinserted polynucleotides into a host cell. Examples of gene deliveryvehicles are liposomes, micelles biocompatible polymers, includingnatural polymers and synthetic polymers; lipoproteins; polypeptides;polysaccharides; lipopolysaccharides; artificial viral envelopes; metalparticles; and bacteria, or viruses, such as baculovirus, adenovirus andretrovirus, bacteriophage, cosmid, plasmid, fungal vectors and otherrecombination vehicles typically used in the art which have beendescribed for expression in a variety of eukaryotic and prokaryotichosts, and may be used for gene therapy as well as for simple proteinexpression.

A polynucleotide of this invention can be delivered to a cell or tissueusing a gene delivery vehicle. “Gene delivery,” “gene transfer,”“transducing,” and the like as used herein, are terms referring to theintroduction of an exogenous polynucleotide (sometimes referred to as a“transgene”) into a host cell, irrespective of the method used for theintroduction. Such methods include a variety of well-known techniquessuch as vector-mediated gene transfer (by, e.g., viralinfection/transfection, or various other protein-based or lipid-basedgene delivery complexes) as well as techniques facilitating the deliveryof “naked” polynucleotides (such as electroporation, “gene gun” deliveryand various other techniques used for the introduction ofpolynucleotides). The introduced polynucleotide may be stably ortransiently maintained in the host cell. Stable maintenance typicallyrequires that the introduced polynucleotide either contains an origin ofreplication compatible with the host cell or integrates into a repliconof the host cell such as an extrachromosomal replicon (e.g., a plasmid)or a nuclear or mitochondrial chromosome. A number of vectors are knownto be capable of mediating transfer of genes to mammalian cells, as isknown in the art and described herein.

A “viral vector” is defined as a recombinantly produced virus or viralparticle that comprises a polynucleotide to be delivered into a hostcell, either in vivo, ex vivo or in vitro. Examples of viral vectorsinclude retroviral vectors, adenovirus vectors, adeno-associated virusvectors, alphavirus vectors and the like. Alphavirus vectors, such asSemliki Forest virus-based vectors and Sindbis virus-based vectors, havealso been developed for use in gene therapy and immunotherapy. See,Schlesinger and Dubensky (1999) Curr. Opin. Biotechnol. 5:434-439 andYing, et al. (1999) Nat. Med. 5(7):823-827. In aspects where genetransfer is mediated by a retroviral vector, a vector construct refersto the polynucleotide comprising the retroviral genome or part thereof,and a therapeutic gene. As used herein, “retroviral mediated genetransfer” or “retroviral transduction” carries the same meaning andrefers to the process by which a gene or nucleic acid sequences arestably transferred into the host cell by virtue of the virus enteringthe cell and integrating its genome into the host cell genome. The viruscan enter the host cell via its normal mechanism of infection or bemodified such that it binds to a different host cell surface receptor orligand to enter the cell. As used herein, retroviral vector refers to aviral particle capable of introducing exogenous nucleic acid into a cellthrough a viral or viral-like entry mechanism.

Retroviruses carry their genetic information in the form of RNA;however, once the virus infects a cell, the RNA is reverse-transcribedinto the DNA form which integrates into the genomic DNA of the infectedcell. The integrated DNA form is called a provirus.

In aspects where gene transfer is mediated by a DNA viral vector, suchas an adenovirus (Ad) or adeno-associated virus (AAV), a vectorconstruct refers to the polynucleotide comprising the viral genome orpart thereof, and a transgene. Adenoviruses (Ads) are a relatively wellcharacterized, homogenous group of viruses, including over 50 serotypes.See, e.g., International PCT Application No. WO 95/27071. Ads do notrequire integration into the host cell genome. Recombinant Ad derivedvectors, particularly those that reduce the potential for recombinationand generation of wild-type virus, have also been constructed. See,International PCT Application Nos. WO 95/00655 and WO 95/11984.Wild-type AAV has high infectivity and specificity integrating into thehost cell's genome. See, Hermonat and Muzyczka (1984) Proc. Natl. Acad.Sci. USA 81:6466-6470 and Lebkowski et al. (1988) Mol. Cell. Biol.8:3988-3996.

Vectors that contain both a promoter and a cloning site into which apolynucleotide can be operatively linked are well known in the art. Suchvectors are capable of transcribing RNA in vitro or in vivo, and arecommercially available from sources such as Stratagene (La Jolla,Calif.) and Promega Biotech (Madison, Wis.). In order to optimizeexpression and/or in vitro transcription, it may be necessary to remove,add or alter 5′ and/or 3′ untranslated portions of the clones toeliminate extra, potential inappropriate alternative translationinitiation codons or other sequences that may interfere with or reduceexpression, either at the level of transcription or translation.Alternatively, consensus ribosome binding sites can be insertedimmediately 5′ of the start codon to enhance expression.

Gene delivery vehicles also include DNA/liposome complexes, micelles andtargeted viral protein-DNA complexes. Liposomes that also comprise atargeting antibody or fragment thereof can be used in the methods ofthis invention. To enhance delivery to a cell, the nucleic acid orproteins of this invention can be conjugated to antibodies or bindingfragments thereof which bind cell surface antigens, e.g., a cell surfacemarker found on stem cells or cardiomyocytes. In addition to thedelivery of polynucleotides to a cell or cell population, directintroduction of the proteins described herein to the cell or cellpopulation can be done by the non-limiting technique of proteintransfection, alternatively culturing conditions that can enhance theexpression and/or promote the activity of the proteins of this inventionare other non-limiting techniques.

The phrase “solid support” refers to non-aqueous surfaces such as“culture plates” “gene chips” or “microarrays.” Such gene chips ormicroarrays can be used for diagnostic and therapeutic purposes by anumber of techniques known to one of skill in the art. In one technique,oligonucleotides are arrayed on a gene chip for determining the DNAsequence by the hybridization approach, such as that outlined in U.S.Pat. Nos. 6,025,136 and 6,018,041. The polynucleotides of this inventioncan be modified to probes, which in turn can be used for detection of agenetic sequence. Such techniques have been described, for example, inU.S. Pat. Nos. 5,968,740 and 5,858,659. A probe also can be affixed toan electrode surface for the electrochemical detection of nucleic acidsequences such as described by Kayem et al. U.S. Pat. No. 5,952,172 andby Kelley et al. (1999) Nucleic Acids Res. 27:4830-4837.

Various “gene chips” or “microarrays” and similar technologies are knowin the art. Examples of such include, but are not limited to, LabCard(ACLARA Bio Sciences Inc.); GeneChip (Affymetrix, Inc); LabChip (CaliperTechnologies Corp); a low-density array with electrochemical sensing(Clinical Micro Sensors); LabCD System (Gamera Bioscience Corp.); OmniGrid (Gene Machines); Q Array (Genetix Ltd.); a high-throughput,automated mass spectrometry systems with liquid-phase expressiontechnology (Gene Trace Systems, Inc.); a thermal jet spotting system(Hewlett Packard Company); Hyseq HyChip (Hyseq, Inc.); BeadArray(Illumina, Inc.); GEM (Incyte Microarray Systems); a high-throughputmicroarrying system that can dispense from 12 to 64 spots onto multipleglass slides (Intelligent Bio-Instruments); Molecular BiologyWorkstation and NanoChip (Nanogen, Inc.); a microfluidic glass chip(Orchid biosciences, Inc.); BioChip Arrayer with four PiezoTippiezoelectric drop-on-demand tips (Packard Instruments, Inc.); FlexJet(Rosetta Inpharmatic, Inc.); MALDI-TOF mass spectrometer (Sequnome);ChipMaker 2 and ChipMaker 3 (TeleChem International, Inc.); andGenoSensor (Vysis, Inc.) as identified and described in Heller (2002)Annu. Rev. Biomed. Eng. 4:129-153. Examples of “gene chips” or a“microarrays” are also described in U.S. Patent Publ. Nos.:2007-0111322, 2007-0099198, 2007-0084997, 2007-0059769 and 2007-0059765and U.S. Pat. Nos. 7,138,506, 7,070,740, and 6,989,267.

In one aspect, “gene chips” or “microarrays” containing probes orprimers homologous to a polynucleotide, polypeptide or antibodydescribed herein are prepared. A suitable sample is obtained from thepatient, extraction of genomic DNA, RNA, protein or any combinationthereof is conducted and amplified if necessary. The sample is contactedto the gene chip or microarray panel under conditions suitable forhybridization of the gene(s) or gene product(s) of interest to theprobe(s) or primer(s) contained on the gene chip or microarray. Theprobes or primers may be detectably labeled thereby identifying thegene(s) of interest. Alternatively, a chemical or biological reactionmay be used to identify the probes or primers which hybridized with theDNA or RNA of the gene(s) of interest. The genotypes or phenotype of thepatient is then determined with the aid of the aforementioned apparatusand methods.

Other non-limiting examples of a solid phase support include glass,polystyrene, polypropylene, polyethylene, dextran, nylon, amylases,natural and modified celluloses, polyacrylamides, gabbros, andmagnetite. The nature of the carrier can be either soluble to someextent or insoluble. The support material may have virtually anypossible structural configuration so long as the coupled molecule iscapable of binding to a polynucleotide, polypeptide or antibody. Thus,the support configuration may be spherical, as in a bead, orcylindrical, as in the inside surface of a test tube, or the externalsurface of a rod. Alternatively, the surface may be flat such as asheet, test strip, etc. or alternatively polystyrene beads. Thoseskilled in the art will know many other suitable carriers for bindingantibody or antigen, or will be able to ascertain the same by use ofroutine experimentation.

“Eukaryotic cells” comprise all of the life kingdoms except monera. Theycan be easily distinguished through a membrane-bound nucleus. Animals,plants, fungi, and protists are eukaryotes or organisms whose cells areorganized into complex structures by internal membranes and acytoskeleton. The most characteristic membrane-bound structure is thenucleus. A eukaryotic host, including, for example, yeast, higher plant,insect and mammalian cells, or alternatively from a prokaryotic cells asdescribed above. Non-limiting examples include simian, bovine, porcine,murine, rats, avian, reptilian and human.

“Prokaryotic cells” that usually lack a nucleus or any othermembrane-bound organelles and are divided into two domains, bacteria andarchaea. Additionally, instead of having chromosomal DNA, these cells'genetic information is in a circular loop called a plasmid. Bacterialcells are very small, roughly the size of an animal mitochondrion (about1-2 μm in diameter and 10 μm long). Prokaryotic cells feature threemajor shapes: rod shaped, spherical, and spiral. Instead of goingthrough elaborate replication processes like eukaryotes, bacterial cellsdivide by binary fission. Examples include but are not limited tobacillus bacteria, E. coli bacterium, and Salmonella bacterium.

The term “human antibody” as used herein, is intended to includeantibodies having variable and constant regions derived from humangermline immunoglobulin sequences. The human antibodies of the inventionmay include amino acid residues not encoded by human germlineimmunoglobulin sequences (e.g., mutations introduced by random orsite-specific mutagenesis in vitro or by somatic mutation in vivo).However, the term “human antibody” as used herein, is not intended toinclude antibodies in which CDR sequences derived from the germline ofanother mammalian species, such as a mouse, have been grafted onto humanframework sequences. Thus, as used herein, the term “human antibody”refers to an antibody in which substantially every part of the protein(e.g., CDR, framework, C_(L), C_(H) domains (e.g., C_(H1), C_(H2),C_(H3)), hinge, (VL, VH)) is substantially non-immunogenic in humans,with only minor sequence changes or variations. Similarly, antibodiesdesignated primate (monkey, baboon, chimpanzee, etc.), rodent (mouse,rat, rabbit, guinea pig, hamster, and the like) and other mammalsdesignate such species, sub-genus, genus, sub-family, family specificantibodies. Further, chimeric antibodies include any combination of theabove. Such changes or variations optionally and preferably retain orreduce the immunogenicity in humans or other species relative tonon-modified antibodies. Thus, a human antibody is distinct from achimeric or humanized antibody. It is pointed out that a human antibodycan be produced by a non-human animal or prokaryotic or eukaryotic cellthat is capable of expressing functionally rearranged humanimmunoglobulin (e.g., heavy chain and/or light chain) genes. Further,when a human antibody is a single chain antibody, it can comprise alinker peptide that is not found in native human antibodies. Forexample, an Fv can comprise a linker peptide, such as two to about eightglycine or other amino acid residues, which connects the variable regionof the heavy chain and the variable region of the light chain. Suchlinker peptides are considered to be of human origin.

As used herein, a human antibody is “derived from” a particular germlinesequence if the antibody is obtained from a system using humanimmunoglobulin sequences, e.g., by immunizing a transgenic mousecarrying human immunoglobulin genes or by screening a humanimmunoglobulin gene library. A human antibody that is “derived from” ahuman germline immunoglobulin sequence can be identified as such bycomparing the amino acid sequence of the human antibody to the aminoacid sequence of human germline immunoglobulins. A selected humanantibody typically is at least 90% identical in amino acids sequence toan amino acid sequence encoded by a human germline immunoglobulin geneand contains amino acid residues that identify the human antibody asbeing human when compared to the germline immunoglobulin amino acidsequences of other species (e.g., murine germline sequences). In certaincases, a human antibody may be at least 95%, or even at least 96%, 97%,98%, or 99% identical in amino acid sequence to the amino acid sequenceencoded by the germline immunoglobulin gene. Typically, a human antibodyderived from a particular human germline sequence will display no morethan 10 amino acid differences from the amino acid sequence encoded bythe human germline immunoglobulin gene. In certain cases, the humanantibody may display no more than 5, or even no more than 4, 3, 2, or 1amino acid difference from the amino acid sequence encoded by thegermline immunoglobulin gene.

A “human monoclonal antibody” refers to antibodies displaying a singlebinding specificity which have variable and constant regions derivedfrom human germline immunoglobulin sequences. The term also intendsrecombinant human antibodies. Methods to making these antibodies aredescribed herein.

The term “recombinant human antibody”, as used herein, includes allhuman antibodies that are prepared, expressed, created or isolated byrecombinant means, such as antibodies isolated from an animal (e.g., amouse) that is transgenic or transchromosomal for human immunoglobulingenes or a hybridoma prepared therefrom, antibodies isolated from a hostcell transformed to express the antibody, e.g., from a transfectoma,antibodies isolated from a recombinant, combinatorial human antibodylibrary, and antibodies prepared, expressed, created or isolated by anyother means that involve splicing of human immunoglobulin gene sequencesto other DNA sequences. Such recombinant human antibodies have variableand constant regions derived from human germline immunoglobulinsequences. In certain embodiments, however, such recombinant humanantibodies can be subjected to in vitro mutagenesis (or, when an animaltransgenic for human Ig sequences is used, in vivo somatic mutagenesis)and thus the amino acid sequences of the VH and VL regions of therecombinant antibodies are sequences that, while derived from andrelated to human germline VH and VL sequences, may not naturally existwithin the human antibody germline repertoire in vivo. Methods to makingthese antibodies are described herein.

As used herein, “isotype” refers to the antibody class (e.g., IgM orIgG1) that is encoded by heavy chain constant region genes.

The terms “polyclonal antibody” or “polyclonal antibody composition” asused herein refer to a preparation of antibodies that are derived fromdifferent B-cell lines. They are a mixture of immunoglobulin moleculessecreted against a specific antigen, each recognizing a differentepitope.

The terms “monoclonal antibody” or “monoclonal antibody composition” asused herein refer to a preparation of antibody molecules of singlemolecular composition. A monoclonal antibody composition displays asingle binding specificity and affinity for a particular epitope.

As used herein, the term “label” intends a directly or indirectlydetectable compound or composition that is conjugated directly orindirectly to the composition to be detected, e.g., polynucleotide orprotein such as an antibody so as to generate a “labeled” composition.The term also includes sequences conjugated to the polynucleotide thatwill provide a signal upon expression of the inserted sequences, such asgreen fluorescent protein (GFP) and the like. The label may bedetectable by itself (e.g. radioisotope labels or fluorescent labels)or, in the case of an enzymatic label, may catalyze chemical alterationof a substrate compound or composition which is detectable. The labelscan be suitable for small scale detection or more suitable forhigh-throughput screening. As such, suitable labels include, but are notlimited to radioisotopes, fluorochromes, chemiluminescent compounds,dyes, and proteins, including enzymes. The label may be simply detectedor it may be quantified. A response that is simply detected generallycomprises a response whose existence merely is confirmed, whereas aresponse that is quantified generally comprises a response having aquantifiable (e.g., numerically reportable) value such as an intensity,polarization, and/or other property. In luminescence or fluorescenceassays, the detectable response may be generated directly using aluminophore or fluorophore associated with an assay component actuallyinvolved in binding, or indirectly using a luminophore or fluorophoreassociated with another (e.g., reporter or indicator) component.

Examples of luminescent labels that produce signals include, but are notlimited to bioluminescence and chemiluminescence. Detectableluminescence response generally comprises a change in, or an occurrenceof, a luminescence signal. Suitable methods and luminophores forluminescently labeling assay components are known in the art anddescribed for example in Haugland, Richard P. (1996) Handbook ofFluorescent Probes and Research Chemicals (6^(th) ed.). Examples ofluminescent probes include, but are not limited to, aequorin andluciferases.

Examples of suitable fluorescent labels include, but are not limited to,fluorescein, rhodamine, tetramethylrhodamine, eosin, erythrosin,coumarin, methyl-coumarins, pyrene, Malacite green, stilbene, LuciferYellow, Cascade Blue™, and Texas Red. Other suitable optical dyes aredescribed in the Haugland, Richard P. (1996) Handbook of FluorescentProbes and Research Chemicals (6^(th) ed.).

In another aspect, the fluorescent label is functionalized to facilitatecovalent attachment to a cellular component present in or on the surfaceof the cell or tissue such as a cell surface marker. Suitable functionalgroups, including, but not are limited to, isothiocyanate groups, aminogroups, haloacetyl groups, maleimides, succinimidyl esters, and sulfonylhalides, all of which may be used to attach the fluorescent label to asecond molecule. The choice of the functional group of the fluorescentlabel will depend on the site of attachment to either a linker, theagent, the marker, or the second labeling agent.

II. Antidotes Factor X and Factor Xa Derivatives

One aspect of the present invention is the use of fX derivatives, suchas Gla-domain deficient fX or fXa or des-Gla fX or fXa, as safe andeffective antidotes to substantially neutralize the activity of aninhibitor of the coagulation fXa to prevent or stop bleeding. It iscontemplated that the antidotes of the present invention will be usefulin reversing the anticoagulant effect of a fXa inhibitor, especially anactive site-directed small molecule inhibitor.

It is contemplated that an antidote to a fXa inhibitor has reduced or noprocoagulant activity but is capable of binding with a fXa inhibitor. Itis contemplated that such limited activity permits dosing of theantidote at a level greater than the circulating wild-type fXa. CertainfXa derivatives, such as des-Gla fXa and Gla-deficient fXa, are suitableantidotes of this invention. Besides having reduced or diminishedprocoagulant activity, antidotes of the present invention should also besubstantially non-immunogenic to the subject. An antidote may contain acombination of two or more the above mutations and/or modifications. Inaddition, any of the above fXa derivatives may be administered alone orin combination with one another.

Factor Xa is a serine protease in the blood coagulation pathwayresponsible for converting prothrombin to thrombin. It is produced fromthe inactive factor X upon activation by either the intrinsic Xase(complex formed by factor IXa with its cofactor, factor VIIIa) or theextrinsic Xase (complex formed by factor VIIa with its cofactor, tissuefactor). Activated fX (fXa) may undergo further autocatalytic cleavageat the C-terminal of its heavy chain, converting fXaα to the subformfXaβ (Jesty, J et al. J. Biol. Chem. 1975, 250(12):4497-4504). Both fXaαand fXaβ are suitable materials for the present invention. fXa itselfconverts prothrombin at slow rate that is not sufficient for supportingcoagulation. Only when it forms a prothrombinase complex with cofactorsCa²⁺, phospholipid, and factor Va, fXa can activate prothrombin at arate rapid enough to support coagulation (Skogen, W. F., et al., J.Biol. Chem. 1984, 259(4):2306-10). The complex requires binding betweenthe negatively charged phospholipid and γ-carboxyglutamic acid residuesin the Gla domain of fXa via Ca²⁺ bridging.

Therefore, although the Gla domain does not contain the active site offXa, it enables fXa to form the prothrombinase complex through theγ-carboxyglutamic acid residues. This is demonstrated by selectiveremoval of fXa Gla-domain by chymotrypsin digestion (see FIG. 7 andExample 1). Clotting assays were performed on fXa during the time courseof cleavage of the Gla domain by chymotrypsin digestion. It has beenreported (Skogen et al J. Biol. Chem. 1984, 259(4):2306-10) that areconstituted prothrombinase complex comprising of Gla-domainless fXa,fVa, phospholipids and calcium ions produces thrombin at a significantlyreduced rate (0.5% product generated compared to control complexcontaining native fXa). As shown in FIG. 7, fXa's activity in clotformation was partially reduced after the fXa was digested bychymotrypsin for 15 minutes and the activity was completely lost after30 minute of digestion. Undercarboxylated or decarboxylated fXa, whichlack the appropriate gamma-carboxyglutamic acid residues required forcalcium ion dependent membrane binding, have thus been found to beincapable of membrane dependent coagulation complex assembly and notsupport blood clotting (Mann, K G et al, Blood, 1990, 76: 1-16).

It has also been established that Gla-domain deficient fXa is capable ofbinding active site-directed inhibitors of fXa. (Brandstetter, H et al,J. Bio. Chem., 1996, 271:29988-29992). There have been reports ofcrystallography of small molecule fXa inhibitor bound to des-Gla humanfXa, which have provided structural description of the active site cleft(Brandstetter, J. Bio. Chem., 1996, 271:29988-29992 and Roehrig, J. Med.Chem. 2005, 48(19):5900-8). FIG. 8 shows that a des-Gla anhydro-fXaexhibited a binding affinity of 0.319 nM with a fXa inhibitorbetrixaban, comparable to that of a native fXa.

It has now been discovered that des-Gla fXa, and other fX derivativesthat have reduced procoagulant activity but are capable of fXa inhibitorbinding, can be used as an antidote to a fXa inhibitor. As shown in FIG.9, the des-Gla anhydro-fXa exhibited complete reversion of betrixaban'santicoagulant activity at a concentration of 680 nM. As detailed inExample 2, the thrombin generation was initiated by adding TF-containingreagent (Innovin) and, thus, indicative of coagulation factors functionin the extrinsic coagulation pathway. It has also been demonstrated inExamples 9-13, that the recombinant antidote is useful to reverse a widevariety of anticoagulants.

Clotting prolongation assays with the activated partial thromboplastintime (aPTT) reagent (Actin FS) that determine the function of thecoagulation factor in the intrinsic coagulation pathway also indicatethat the des-Gla anhydro-fXa possess antidote activity. FIG. 10 showsthe dose responsive antidote effect of des-Gla anhydro-fXa against 250nM of betrixaban, with complete reversion at 600 nM. FIG. 11 shows thatdes-Gla anhydro-fXa was also capable of reversing the anticoagulantactivity of another fXa inhibitor, enoxaparin. FIG. 12 shows thatdes-Gla anhydro-fXa did not exhibit significant antidote activityagainst a direct thrombin inhibitor argatroban. Thus, the des-Glaanhydro-fXa is a selective antidote for fXa inhibitors and is capable ofrestoring fXa procoagulant activity initiated either by the extrinsic orthe intrinsic pathway.

Further, the antidote activity of des-Gla anhydro-fXa was demonstratedby the aPTT prolongation assays measured with a traditional coagulationtimer. As shown in FIG. 13, des-Gla anhydro-fXa itself has no effect onaPTT of control plasma at the highest concentrations tested (2576 nM).400 nM of betrixaban extended aPTT more than two folds. Thisanti-coagulant effect of betrixaban is reversed by des-Gla anhydro-fXain a dose-responsive manner, with return of aPTT to near normal level ofcontrol plasma at antidote concentrations higher than 1610 nM.

It is contemplated that further truncations at the fXa light chain, forexample, additional deletion of the EGF1 domain, EGF1 plus EGF2 domains,or fragments thereof, and inactive fXa with only the heavy chain may beuseful antidotes of this invention. In one embodiment, the derivativecomprises deletion of substantially all of the EGF1 domain, deletion ofsubstantially all of an EGF2 domains, or both. In one embodiment,deletion of substantially all of the EGF1 domain comprises at leastabout 50% or least about 80% of amino acid residues 46 to 84 of SEQ IDNO. 3 or an equivalent thereof. In another embodiment, deletion ofsubstantially all of the EGF2 domain comprises at least about 50% or atleast about 80% of amino acid residues 85-128 of SEQ ID NO. 3.

Gla-domain deficient fXa does not support normal coagulation underphysiologically relevant concentration. However, the protein has theability of cleaving many substrates and causing clotting at higherconcentrations. For example, Skogen et al (Skogen, W. F., et al., J.Biol. Chem. 1984, 259(4):2306-10) showed that bovine des-Gla fXa hasabout 0.5-1.0% prothrombinase complex activity relative to the wild typefXa. Thus, modifications that further reduce or completely eliminate afXa derivative's procoagulant activity is contemplated by methods of theinvention. Such modification may be, for example, in a fXa's catalyticdomain.

Several ways of modifying the catalytic domain in the fXa heavy chain toreduce its procoagulant activity are contemplated. The active siteresidue 5379 of fXa (as shown in SEQ ID NO. 7), for example, can beselectively replaced by dehydro-alanine (see Example 1) or alanine (seeExample 6) to reduce or eliminate the procoagulant activity. It is alsoknown that complex formation between fXa and a reagent targeting fXa'sexosite may block the macromolecular binding ability of fXa, thusreducing its procoagulant activity while retaining small moleculebinding ability in the active site. This exosite targeting reagentincludes, without limitation, monoclonal antibodies targeting a regionremoved from the active site (Wilkens, M and Krishnaswamy, S, J. Bio.Chem., 2002, 277 (11), 9366-9374), or α-2-macroglobulin. It has beenknown that the α-2-macroglobulin-serine protease complex, such as withtrypsin, thrombin or fXa, is capable of binding small moleculesubstrates (Kurolwa, K. et al, Clin. Chem. 1989, 35(11), 2169-2172).

It is also known that an inactive fXa with modifications solely in theheavy chain while keeping its light chain unchanged would act as aninhibitor of prothrombinase (Hollenbach, S. et al., Thromb. Haemost.,1994, 71(3), 357-62) because it interferes with procoagulant activity ofnormal fXa as shown in FIG. 6. Therefore, in one embodiment, the fXaderivative has modifications both in the light chain and heavy chain. Ithas been discovered that these modifications reduce or eliminate bothprocoagulant and anticoagulant activities while retaining the inhibitorsbinding ability of the fXa derivative.

Several methods can be used to produce Gla-domain deficient fXaderivatives or other fXa derivatives described herein. For example, theGla-domain may be completely removed via chymotryptic cleavage,producing Gla-domainless fXa. Alternatively, a Gla-domainless fX may beproduced by chymotryptic cleavage of native fX. The Gla-domainless fXmay then be converted to Gla-domainless fXa by a fX activator. fX may beisolated from plasma of the same or a different species as the subjectto be treated. Bovine fX, for example, has been shown to be functionalin human plasma assays. Examples of a fX activator include, withoutlimitation, a snake venom, such as Russell's viper venom, and complexesof fVIIa/tissue factor or fIXa/fVIIIa. Such means is known to a personof skill in the art. For example, Rudolph A. E. et al has reported arecombinant fXa produced from a recombinant factor X (fX) with a singlesubstitution of Arg347 by Glutamine (fXR347N) (Biochem. 2000, 39 (11):2861-2867). In one embodiment, the fXa derivatives produced fromnon-human sources are non-immunogenic or substantially non-immunogenic.Example 7 also provides a method of producing a recombinant antidotehaving the amino acid sequence of SEQ ID NO. 12.

The fXa derivatives may also be purified from human plasma, or may beproduced by recombinant DNA method where an appropriate gene for the fXaderivative is expressed in a suitable host organism. Expression andpurification of recombinant fXa has been reported by several groups,see, e.g., Larson, P. J., et al, Biochem., 1998, 37:5029-5038, andCamire, R. M., et al, Biochem., 2000, 39, 14322-14329 for producingrecombinant IX; Wolf, D. L. et al, J. Bio. Chem., 1991,266(21):13726-13730 for producing recombinant fXa. Modified fXa may beprepared according to these procedures using a genetically modified cDNAhaving a nucleotide sequence encoding the desired fXa mutant. Example 6gives more details for direct expression of a Gla-domainless fXa-S379mutant with functional activity as an antidote.

It is contemplated that active-site mutated or modified fXa withdeficient Gla-domain, such as under-carboxylated fXa, may also be usefulas fXa inhibitor antidote. Under-carboxylated fXa may be prepared byrecombinant means by withholding vitamin K derivatives during proteinexpression (vitamin K derivatives are needed for post translationalmodification to form the Gla residues) or by adding vitamin Kantagonists such as warfarin during tissue culture. Decarboxylated fXacan be prepared by heating (Bajaj P., J. Biol. Chem., 1982,257(7):3726-3731) or by proteolytic digestion by chymotrypsin (MoritaT., et al., J. Biol. Chem., 1986, 261(9):4015-4023). The antidote mayalso be generated in prokaryotic systems followed by in vitro refoldingor constitution of the fXa inhibitor binding site.

The Gla residues can also be chemically modified to remove the carboxylgroup responsible for calcium ion dependent membrane binding. Forexample, the carboxyl groups on the Gla residues may be selectivelyremoved under decarboxylation conditions or may be capped, for example,by esterification or amination. It is desirable that such esterificationor amination be resistant to in vivo hydrolysis so that the modified fXais not readily converted to active fXa, which may cause thrombosis.

Other mutants or derivatives of fXa may also be useful antidotes of thisinvention. In one embodiment, this invention encompasses use of mutantsdescribed in Peter J. Larson et al, Biochem., 1998, 37:5029-5038 as fXainhibitor antidotes.

Additional modifications of the fX protein may be made to produce usefulantidotes to fXa inhibitors. One such modification includes deleting allor part of the activation peptide at the N-terminus of the heavy chain.In one embodiment, this includes deletion of all of part of theactivation peptide or amino acid residues 143-194 of SEQ ID NO. 3 or anequivalent thereof. This may be accomplished by procedures described inSinha et al. Protein Expression and Purification, 3, 518-524 (1992) andWolf et al. J. Biol. Chem. 266(21), 13726-13730 (1991), which are bothincorporated herein by reference. Additional methods are describedbelow.

In another embodiment, an amino acid modification prevents cleavage of a13-peptide wherein (3-peptide refers to a portion of or the entire heavychain. This modification can include deletion, substitution, orinsertion of an amino acid. On such modification includes substitutionsof one or more of Arg429 or Ser436. For example, there is a Arg 429Glnsubstitution and may also include a Thr443Ala substitution. Conversionof fXaα to fXaβ has been described by, for example, Pryzdial and Kessler(Pryzdial L G and Kessler G E, Journal of Biological Chemistry, 271(28),16621-16626 (1996). U.S. Pat. No. 7,220,569(B2) described modificationsin the fX activation peptide and β-peptide. This reference isincorporated by reference in its entirety.

In still another embodiment, the derivative comprises a deletion of aβ-peptide. In one embodiment, the deletion of the β-peptide comprises adeletion of at least amino acid residues 430-448 of SEQ ID NO. 3 or anequivalent thereof.

In another embodiment, a native factor X signal peptide sequence or analternative signal peptide sequence is used to express the derivatives.For example, the native factor X signal peptide (SEQ ID NO. 18) can beused as the signal sequence for SEQ ID NO. 12-15 and 19-25. The signalpeptide may be selected from a factor X signal peptide (SEQ ID NO. 18),a transferrin signal peptide (amino acid residues SP1-SP19 of SEQ ID NO.27) and a prothrombin signal peptide (amino acid residues SP1-SP24 ofSEQ ID NO. 26). It is contemplated that the factor X signal peptide maybe used in as the signal peptide of any of the sequences describedherein. Use of prothrombin signal peptide in replacing native fX signalpeptide has been described by Guarna et al (Guarna M M et al, ProteinExpression and Purification, 20(2) 133-41 (2000)) and Camire et al(Camire R M, et al, Biochemistry, 39, 14322-14329 (2000)). Use oftransferring signal peptide has been described by Rezaie and Esmon(Rezaie A R and Charles T. Esmon C T, J. Biol. Chem., 267(36),26104-26109 (1992)). Both references are incorporated by reference intheir entirety.

In yet another embodiment of the invention, the polypeptide has aprecursor peptide which is a one chain polypeptide. In one embodiment,there is a RKR (SEQ ID NO. 40) linker at a C-terminus of a light chainor a RKRRKR (SEQ ID NO. 41) linker at the C-terminus of a light chain.In another embodiment, the derivative is a two-chain polypeptide.Expression of fXa mutant suitable as antidote or antidote precursor ofthe invention with RKRRKR linker (SEQ ID NO: 41) has been fullydescribed in US patent publication 2009/0098119A1. Use of RKR (SEQ IDNO. 40) linker to connect fXa light chain and activation peptiden hasbeen 49 discussed in U.S. Pat. No. 5,990,079. Both the patent and thepatent application just listed are incorporated by reference in theirentirety.

In another embodiment, this invention encompasses use of catalyticallyinactive fXa mutants to prepare fXa inhibitor antidotes. For example,mutants described in Sinha, U., et al, Protein Expression and Purif.,1992, 3:518-524 rXai, mutants with chemical modifications, such asdehydro-alanine (anhydro fXa), as described in Nogami, et al, J. Biol.Chem. 1999, 274(43):31000-7. FXa with active site serine (Ser379 in fXnumbering as shown in SEQ ID NO. 7, and Ser195 in chymotrypsinnumbering) replaced with alanine (fXa-S379A in fX numbering, orfXa-S195A in chymotrypsin numbering), where the procoagulant activitywas eliminated, may also be used as fXa inhibitor antidotes. Theinvention also envisions fXa derivatives with the active site serineresidue irreversibly acylated which is still capable of binding smallmolecule inhibitors. FXa with the active site serine reversibly acylatedhas been reported by Wolf, et al., Blood, 1995, 86(11):4153-7. Suchreversible acylation, however, is capable of time dependent productionof active fXa and may lead to an excess of active fXa over a timeperiod. The deacylation rate may be reduced by strategies similar tothose described in Lin P. H. et al, Thrombosis Res., 1997, 88(4),365-372. For example, fXa molecules with Ser379 (Ser195 in chymotrypsinnumbering) acylated by 4-methoxybenzyl and 3-bromo-4-methoxybenzylgroups recover less than 50% of their original activity when incubatedin a buffer having pH 7.5 at 37° C. for 4 hours.

One embodiment is directed to the use of fXa derivatives with mutationsat fXa residues known to be important for fXa interaction with cofactorfV/fVa. Such residues include, without limitation, Arg306, Glu310,Arg347, Lys351, or Lys414 (SEQ ID NOS. 3 and 7, these amino acidscorrespond to Arg125, Glu129, Arg165, Lys169, Lys230 in the chymotrypsinnumbering). Examples of such mutants are reported in Rudolph, A. E. etal, J. Bio. Chem., 2001, 276:5123-5128. In addition, mutations at fXaresidues known to be important for fVIII/fVIIIa interaction, such asArg424 in SEQ ID NOS. 3 and 7 (Arg240 in chymotrypsin numbering), mayalso be used as fXa inhibitor antidotes. Examples of such mutants aredescribed in Nogami, K. et al, J. Biol. Chem., 2004,279(32):33104-33113.

Other modification of active site residues of fXa or residues known tobe important for serine protease interactions may also lead to usefulantidotes of this invention, for example, replacement of Glu216, Glu218,and Arg332 in SEQ ID NOS. 3 and 7 (Glu37, Glu39, and Arg 150 inchymotrypsin numbering, respectively) with other amino acid residues.

One embodiment is directed to mutations at the autolysis loop of FXaheavy chain to eliminate potential degradation. FXa, like other serineproteases of the family, has an exposed surface loop (autolysis loop)which is susceptible to cleavage by various proteases. This loop,including amino acids RTHEKGRQSTR (SEQ ID NO. 39), which are amino acids366 to 376 of SEQ ID NO. 1, contains several positively charged residues(Arg366, Lys370, Arg372, Arg376) with Arg372 as the potentialrecognition site for cleavage (Brandstetter H., et. Al., J. Biol. Chem.,271 (1996), 29988-29992). It is completed that these charged residuesare mutated to Gln (Q) or Ala (A) to prohibit the possible cleavage. Inone aspect, the mutation is Arg366Q/A. In another aspect, the mutationis Lys370Q/A. In yet another aspect, the mutation is Arg372Q/A. In yetanother aspect, the mutation is Arg376Q/A. In some embodiments, a fXaderivative has one or more such mutations.

One embodiment of the invention is directed to an isolated polypeptideof SEQ ID NOs. 12, 13, 15, 19-25, or an isolated polypeptide having atleast 80% homology to SEQ ID NOs. 12, 13, 15, 19-25, further comprisingone or more mutations selected from the group consisting of Arg366Q/A,Lys370Q/A, Arg372Q/A, Arg376Q/A, and combinations thereof. Thepolypeptides encompassed by the invention include both the single chainpolypeptide (i.e., before the polypeptide is excreted from the cell) aswell as the two-chain polypeptide (i.e., after cleavage at the linkerafter it is secreted from the cell).

In one embodiment, the residual procoagulant activity of an antidote, asassessed by amidolytic substrate cleavage assay, be <1%, preferably<0.1%, more preferably <0.05% of human plasma derived native fXa. Forexample, there is no measurable procoagulant activity for recombinantfXa-S379A when the active site Ser379 (S195 in chymotrypsin numbering)is replaced by an alanine residue as measured by clotting assays.

The invention further relates to nucleic acid sequences, in particularDNA sequences, which code for the fXa derivatives described above. Thesecan easily be determined by translating the polypeptide sequence backinto the corresponding DNA sequence in accordance with the genetic code.Codons preferably used are those which lead to good expression in therequired host organism. The nucleic acid sequences can be preparedeither by site-specific mutagenesis starting from the natural fXa genesequence or else by complete DNA synthesis.

Polypeptides of the Invention

In certain aspects, the invention is related to an isolated polypeptidecomprising the amino acid sequence of SEQ ID NO. 12, 13, 15, or 19-25.Also encompassed by this invention are polypeptides having at least 80%homology to SEQ ID NO. 12, 13, 15, or 19-25.

Polypeptides comprising the amino acid sequences of the invention can beprepared by expressing polynucleotides encoding the polypeptidesequences of this invention in an appropriate host cell. This can beaccomplished by methods of recombinant DNA technology known to thoseskilled in the art. Accordingly, this invention also provides methodsfor recombinantly producing the polypeptides of this invention in aeukaryotic or prokaryotic host cells. The proteins and polypeptides ofthis invention also can be obtained by chemical synthesis using acommercially available automated peptide synthesizer such as thosemanufactured by Perkin Elmer/Applied Biosystems, Inc., Model 430A or431A, Foster City, Calif., USA. The synthesized protein or polypeptidecan be precipitated and further purified, for example by highperformance liquid chromatography (HPLC). Accordingly, this inventionalso provides a process for chemically synthesizing the proteins of thisinvention by providing the sequence of the protein and reagents, such asamino acids and enzymes and linking together the amino acids in theproper orientation and linear sequence.

It is known to those skilled in the art that modifications can be madeto any peptide to provide it with altered properties. Polypeptides ofthe invention can be modified to include unnatural amino acids. Thus,the peptides may comprise D-amino acids, a combination of D- and L-aminoacids, and various “designer” amino acids (e.g., β-methyl amino acids,C-α-methyl amino acids, and N-α-methyl amino acids, etc.) to conveyspecial properties to peptides. Additionally, by assigning specificamino acids at specific coupling steps, peptides with α-helices, βturns, β sheets, α-turns, and cyclic peptides can be generated.Generally, it is believed that α-helical secondary structure or randomsecondary structure is preferred.

In a further embodiment, subunits of polypeptides that confer usefulchemical and structural properties will be chosen. For example, peptidescomprising D-amino acids may be resistant to L-amino acid-specificproteases in vivo. Modified compounds with D-amino acids may besynthesized with the amino acids aligned in reverse order to produce thepeptides of the invention as retro-inverse peptides. In addition, thepresent invention envisions preparing peptides that have better definedstructural properties, and the use of peptidomimetics, andpeptidomimetic bonds, such as ester bonds, to prepare peptides withnovel properties. In another embodiment, a peptide may be generated thatincorporates a reduced peptide bond, i.e., R₁—CH₂NH—R₂, where R₁, and R₂are amino acid residues or sequences. A reduced peptide bond may beintroduced as a dipeptide subunit. Such a molecule would be resistant topeptide bond hydrolysis, e.g., protease activity. Such molecules wouldprovide ligands with unique function and activity, such as extendedhalf-lives in vivo due to resistance to metabolic breakdown, or proteaseactivity. Furthermore, it is well known that in certain systemsconstrained peptides show enhanced functional activity (Hruby (1982)Life Sciences 31:189-199 and Hruby et al. (1990) Biochem J.268:249-262); the present invention provides a method to produce aconstrained peptide that incorporates random sequences at all otherpositions.

The following non-classical amino acids may be incorporated in thepeptides of the invention in order to introduce particularconformational motifs: 1,2,3,4-tetrahydroisoquinoline-3-carboxylate(Kazrnierski et al. (1991) J. Am. Chem. Soc. 113:2275-2283);(2S,3S)-methyl-phenylalanine, (2S,3R)-methyl-phenylalanine,(2R,3S)-methyl-phenylalanine and (2R,3R)-methyl-phenylalanine(Kazmierski and Hruby (1991) Tetrahedron Lett. 32(41):5769-5772);2-aminotetrahydronaphthalene-2-carboxylic acid (Landis (1989) Ph.D.Thesis, University of Arizona);hydroxy-1,2,3,4-tetrahydroisoquinoline-3-carboxylate (Miyake et al.(1989) J. Takeda Res. Labs. 43:53-76) histidine isoquinoline carboxylicacid (Zechel et al. (1991) Int. J. Pep. Protein Res. 38(2):131-138); andHIC (histidine cyclic urea), (Dharanipragada et al. (1993) Int. J. Pep.Protein Res. 42(1):68-77) and (Dharanipragada et al. (1992) Acta.Crystallogr. C. 48:1239-1241).

The following amino acid analogs and peptidomimetics may be incorporatedinto a peptide to induce or favor specific secondary structures: LL-Acp(LL-3-amino-2-propenidone-6-carboxylic acid), a β-turn inducingdipeptide analog (Kemp et al. (1985) J. Org. Chem. 50:5834-5838);β-sheet inducing analogs (Kemp et al. (1988) Tetrahedron Lett.29:5081-5082); β-turn inducing analogs (Kemp et al. (1988) TetrahedronLett. 29:5057-5060); α-helix inducing analogs (Kemp et al. (1988)Tetrahedron Lett. 29:4935-4938); α-turn inducing analogs (Kemp et al.(1989) J. Org. Chem. 54:109:115); analogs provided by the followingreferences: Nagai and Sato (1985) Tetrahedron Lett. 26:647-650; andDiMaio et al. (1989) J. Chem. Soc. Perkin Trans. p. 1687; a Gly-Ala turnanalog (Kahn et al. (1989) Tetrahedron Lett. 30:2317); amide bondisostere (Clones et al. (1988) Tetrahedron Lett. 29:3853-3856);tetrazole (Zabrocki et al. (1988) J. Am. Chem. Soc. 110:5875-5880); DTC(Samanen et al. (1990) Int. J. Protein Pep. Res. 35:501:509); andanalogs taught in Olson et al. (1990) J. Am. Chem. Sci. 112:323-333 andGarvey et al. (1990) J. Org. Chem. 56:436. Conformationally restrictedmimetics of beta turns and beta bulges, and peptides containing them,are described in U.S. Pat. No. 5,440,013, issued Aug. 8, 1995 to Kahn.

It is known to those skilled in the art that modifications can be madeto any peptide by substituting one or more amino acids with one or morefunctionally equivalent amino acids that does not alter the biologicalfunction of the peptide. In one aspect, the amino acid that issubstituted by an amino acid that possesses similar intrinsic propertiesincluding, but not limited to, hydrophobicity, size, or charge. Methodsused to determine the appropriate amino acid to be substituted and forwhich amino acid are known to one of skill in the art. Non-limitingexamples include empirical substitution models as described by Dayhoffet al. (1978) In Atlas of Protein Sequence and Structure Vol. 5 suppl. 2(ed. M. O. Dayhoff), pp. 345-352. National Biomedical ResearchFoundation, Washington D.C.; PAM matrices including Dayhoff matrices(Dayhoff et al. (1978), supra, or JTT matrices as described by Jones etal. (1992) Comput. Appl. Biosci. 8:275-282 and Gonnet et al. (1992)Science 256:1443-1145; the empirical model described by Adach andHasegawa (1996) J. Mol. Evol. 42:459-468; the block substitutionmatrices (BLOSUM) as described by Henikoff and Henikoff (1992) Proc.Natl. Acad. Sci. USA 89:10915-10919; Poisson models as described by Nei(1987) Molecular Evolutionary Genetics. Columbia University Press, NewYork.; and the Maximum Likelihood (ML) Method as described by Müer etal. (2002) Mol. Biol. Evol. 19:8-13.

Polypeptide Conjugates

The polypeptides and polypeptide complexes of the invention can be usedin a variety of formulations, which may vary depending on the intendeduse. For example, one or more can be covalently or non-covalently linked(complexed) to various other molecules, the nature of which may varydepending on the particular purpose. For example, a peptide of theinvention can be covalently or non-covalently complexed to amacromolecular carrier, including, but not limited to, natural andsynthetic polymers, proteins, polysaccharides, polypeptides (aminoacids), polyvinyl alcohol, polyvinyl pyrrolidone, and lipids. A peptidecan be conjugated to a fatty acid, for introduction into a liposome, seeU.S. Pat. No. 5,837,249. A peptide of the invention can be complexedcovalently or non-covalently with a solid support, a variety of whichare known in the art and described herein. An antigenic peptide epitopeof the invention can be associated with an antigen-presenting matrixsuch as an MHC complex with or without co-stimulatory molecules.

Examples of protein carriers include, but are not limited to,superantigens, serum albumin, tetanus toxoid, ovalbumin, thyroglobulin,myoglobulin, and immunoglobulin.

Peptide-protein carrier polymers may be formed using conventionalcross-linking agents such as carbodiimides. Examples of carbodimides are1-cyclohexyl-3-(2-morpholinyl-(4-ethyl) carbodiimide (CMC),1-ethyl-3-(3-dimethyaminopropyl) carbodiimide (EDC) and1-ethyl-3-(4-azonia-44-dimethylpentyl) carbodiimide.

Examples of other suitable cross-linking agents are cyanogen bromide,glutaraldehyde and succinic anhydride. In general, any of a number ofhomo-bifunctional agents including a homo-bifunctional aldehyde, ahomo-bifunctional epoxide, a homo-bifunctional imido-ester, ahomo-bifunctional N-hydroxysuccinimide ester, a homo-bifunctionalmaleimide, a homo-bifunctional alkyl halide, a homo-bifunctional pyridyldisulfide, a homo-bifunctional aryl halide, a homo-bifunctionalhydrazide, a homo-bifunctional diazonium derivative and ahomo-bifunctional photoreactive compound may be used. Also included arehetero-bifunctional compounds, for example, compounds having anamine-reactive and a sulfhydryl-reactive group, compounds with anamine-reactive and a photoreactive group and compounds with acarbonyl-reactive and a sulfhydryl-reactive group.

Specific examples of such homo-bifunctional cross-linking agents includethe bifunctional N-hydroxysuccinimide estersdithiobis(succinimidylpropionate), disuccinimidyl suberate, anddisuccinimidyl tartrate; the bifunctional imido-esters dimethyladipimidate, dimethyl pimelimidate, and dimethyl suberimidate; thebifunctional sulfhydryl-reactive crosslinkers1,4-di-[3′-(2′-pyridyldithio) propionamido]butane, bismaleimidohexane,and bis-N-maleimido-1,8-octane; the bifunctional aryl halides1,5-difluoro-2,4-dinitrobenzene and4,4′-difluoro-3,3′-dinitrophenylsulfone; bifunctional photoreactiveagents such as bis-[b-(4-azidosalicylamido)ethyl]disulfide; thebifunctional aldehydes formaldehyde, malondialdehyde, succinaldehyde,glutaraldehyde, and adipaldehyde; a bifunctional epoxide such as1,4-butaneodiol diglycidyl ether; the bifunctional hydrazides adipicacid dihydrazide, carbohydrazide, and succinic acid dihydrazide; thebifunctional diazoniums o-tolidine, diazotized and bis-diazotizedbenzidine; the bifunctional alkylhalidesN1N′-ethylene-bis(iodoacetamide), N1N′-hexamethylene-bis(iodoacetamide),N1N′-undecamethylene-bis(iodoacetamide), as well as benzylhalides andhalomustards, such as a1a′-diiodo-p-xylene sulfonic acid andtri(2-chloroethyl)amine, respectively.

Examples of common hetero-bifunctional cross-linking agents that may beused to effect the conjugation of proteins to peptides include, but arenot limited to, SMCC(succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate), MBS(m-maleimidobenzoyl-N-hydroxysuccinimide ester), SIAB(N-succinimidyl(4-iodoacteyl)aminobenzoate), SMPB(succinimidyl-4-(p-maleimidophenyl)butyrate), GMBS(N-(γ-maleimidobutyryloxy)succinimide ester), MPBH(4-(4-N-maleimidopohenyl) butyric acid hydrazide), M2C2H(4-(N-maleimidomethyl)cyclohexane-1-carboxyl-hydrazide), SMPT(succinimidyloxycarbonyl-α-methyl-α-(2-pyridyldithio)toluene), and SPDP(N-succinimidyl 3-(2-pyridyldithio)propionate).

Cross-linking may be accomplished by coupling a carbonyl group to anamine group or to a hydrazide group by reductive amination.

Peptides of the invention also may be formulated as non-covalentattachment of monomers through ionic, adsorptive, or biospecificinteractions. Complexes of peptides with highly positively or negativelycharged molecules may be done through salt bridge formation under lowionic strength environments, such as in deionized water. Large complexescan be created using charged polymers such as poly-(L-glutamic acid) orpoly-(L-lysine) which contain numerous negative and positive charges,respectively. Adsorption of peptides may be done to surfaces such asmicroparticle latex beads or to other hydrophobic polymers, formingnon-covalently associated peptide-superantigen complexes effectivelymimicking cross-linked or chemically polymerized protein. Finally,peptides may be non-covalently linked through the use of biospecificinteractions between other molecules. For instance, utilization of thestrong affinity of biotin for proteins such as avidin or streptavidin ortheir derivatives could be used to form peptide complexes. Thesebiotin-binding proteins contain four binding sites that can interactwith biotin in solution or be covalently attached to another molecule.(See Wilchek (1988) Anal. Biochem. 171:1-32). Peptides can be modifiedto possess biotin groups using common biotinylation reagents such as theN-hydroxysuccinimidyl ester of D-biotin (NHS-biotin) which reacts withavailable amine groups on the protein. Biotinylated peptides then can beincubated with avidin or streptavidin to create large complexes. Themolecular mass of such polymers can be regulated through careful controlof the molar ratio of biotinylated peptide to avidin or streptavidin.

Also provided by this application are the peptides and polypeptidesdescribed herein conjugated to a label, e.g., a fluorescent orbioluminescent label, for use in the diagnostic methods. For example,detectably labeled peptides and polypeptides can be bound to a columnand used for the detection and purification of antibodies. Suitablefluorescent labels include, but are not limited to, fluorescein,rhodamine, tetramethylrhodamine, eosin, erythrosin, coumarin,methyl-coumarins, pyrene, Malacite green, stilbene, Lucifer Yellow,Cascade Blue™, and Texas Red. Other suitable optical dyes are describedin Haugland, Richard P. (1996) Molecular Probes Handbook.

The polypeptides of this invention also can be combined with variousliquid phase carriers, such as sterile or aqueous solutions,pharmaceutically acceptable carriers, suspensions and emulsions.Examples of non-aqueous solvents include propyl ethylene glycol,polyethylene glycol and vegetable oils. When used to prepare antibodies,the carriers also can include an adjuvant that is useful tonon-specifically augment a specific immune response. A skilled artisancan easily determine whether an adjuvant is required and select one.However, for the purpose of illustration only, suitable adjuvantsinclude, but are not limited to, Freund's Complete Adjuvant, Freund'sIncomplete Adjuvant and mineral salts.

Host Cells

Also provided are host cells comprising one or more of the polypeptidesof this invention. In one aspect, the polypeptides are expressed andpresent on the cell surface (extracellularly). Suitable cells containingthe inventive polypeptides include prokaryotic and eukaryotic cells,which include, but are not limited to bacterial cells, yeast cells,insect cells, animal cells, mammalian cells, murine cells, rat cells,sheep cells, simian cells and human cells. Examples of bacterial cellsinclude Escherichia coli, Salmonella enterica and Streptococcusgordonii. The cells can be purchased from a commercial vendor such asthe American Type Culture Collection (ATCC, Rockville Md., USA) orcultured from an isolate using methods known in the art. Examples ofsuitable eukaryotic cells include, but are not limited to 293T HEKcells, as well as the hamster cell line CHO, BHK-21; the murine celllines designated NIH3T3, NS0, C127, the simian cell lines COS, Vero; andthe human cell lines HeLa, PER.C6 (commercially available from Crucell)U-937 and Hep G2. A non-limiting example of insect cells includeSpodoptera frugiperda. Examples of yeast useful for expression include,but are not limited to Saccharomyces, Schizosaccharomyces, Hansenula,Candida, Torulopsis, Yarrowia, or Pichia. See e.g., U.S. Pat. Nos.4,812,405; 4,818,700; 4,929,555; 5,736,383; 5,955,349; 5,888,768 and6,258,559.

In addition to species specificity, the cells can be of any particulartissue type such as neuronal or alternatively a somatic or embryonicstem cell such as a stem cell that can or cannot differentiate into aneuronal cell, e.g., embryonic stem cell, adipose stem cell, neuronalstem cell and hematopoietic stem cell. The stem cell can be of human oranimal origin, such as mammalian.

Isolated Polynucleotides and Compositions

This invention also provides the complementary polynucleotides to thesequences identified above or their complements. Complementarity can bedetermined using traditional hybridization under conditions of moderateor high stringency. As used herein, the term polynucleotide intends DNAand RNA as well as Modified nucleotides. For example, this inventionalso provides the anti-sense polynucleotide stand, e.g. antisense RNA tothese sequences or their complements.

Also provided are polynucleotides encoding substantially homologous andbiologically equivalent polypeptides to the inventive polypeptides andpolypeptide complexes. Substantially homologous and biologicallyequivalent intends those having varying degrees of homology, such as atleast 65%, or alternatively, at least 70%, or alternatively, at least75%, or alternatively at least 80%, or alternatively, at least 85%, oralternatively at least 90%, or alternatively, at least 95%, oralternatively at least 97% homologous as defined above and which encodepolypeptides having the biological activity to bind factor Xa inhibitorsand do not assemble into the prothrombinase complex as described herein.It should be understood although not always explicitly stated thatembodiments to substantially homologous polypeptides and polynucleotidesare intended for each aspect of this invention, e.g., polypeptides,polynucleotides and antibodies.

The polynucleotides of this invention can be replicated usingconventional recombinant techniques. Alternatively, the polynucleotidescan be replicated using PCR technology. PCR is the subject matter ofU.S. Pat. Nos. 4,683,195; 4,800,159; 4,754,065; and 4,683,202 anddescribed in PCR: The Polymerase Chain Reaction (Mullis et al. eds,Birkhauser Press, Boston (1994)) and references cited therein. Yetfurther, one of skill in the art can use the sequences provided hereinand a commercial DNA synthesizer to replicate the DNA. Accordingly, thisinvention also provides a process for obtaining the polynucleotides ofthis invention by providing the linear sequence of the polynucleotide,appropriate primer molecules, chemicals such as enzymes and instructionsfor their replication and chemically replicating or linking thenucleotides in the proper orientation to obtain the polynucleotides. Ina separate embodiment, these polynucleotides are further isolated. Stillfurther, one of skill in the art can operatively link thepolynucleotides to regulatory sequences for their expression in a hostcell. The polynucleotides and regulatory sequences are inserted into thehost cell (prokaryotic or eukaryotic) for replication and amplification.The DNA so amplified can be isolated from the cell by methods well knownto those of skill in the art. A process for obtaining polynucleotides bythis method is further provided herein as well as the polynucleotides soobtained.

RNA can be obtained by first inserting a DNA polynucleotide into asuitable prokaryotic or eukaryotic host cell. The DNA can be inserted byany appropriate method, e.g., by the use of an appropriate gene deliveryvehicle (e.g., liposome, plasmid or vector) or by electroporation. Whenthe cell replicates and the DNA is transcribed into RNA; the RNA canthen be isolated using methods well known to those of skill in the art,for example, as set forth in Sambrook and Russell (2001) supra. Forinstance, mRNA can be isolated using various lytic enzymes or chemicalsolutions according to the procedures set forth in Sambrook and Russell(2001) supra or extracted by nucleic-acid-binding resins following theaccompanying instructions provided by manufactures.

In one aspect, the RNA is short interfering RNA, also known as siRNA.Methods to prepare and screen interfering RNA and select for the abilityto block polynucleotide expression are known in the art and non-limitingexamples of which are shown below. These interfering RNA are provided bythis invention.

siRNA sequences can be designed by obtaining the target mRNA sequenceand determining an appropriate siRNA complementary sequence. siRNAs ofthe invention are designed to interact with a target sequence, meaningthey complement a target sequence sufficiently to hybridize to thatsequence. An siRNA can be 100% identical to the target sequence.However, homology of the siRNA sequence to the target sequence can beless than 100% as long as the siRNA can hybridize to the targetsequence. Thus, for example, the siRNA molecule can be at least 70%,75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to thetarget sequence or the complement of the target sequence. Therefore,siRNA molecules with insertions, deletions or single point mutationsrelative to a target may also be used. The generation of severaldifferent siRNA sequences per target mRNA is recommended to allowscreening for the optimal target sequence. A homology search, such as aBLAST search, should be performed to ensure that the siRNA sequence doesnot contain homology to any known mammalian gene.

In general, it is preferable that the target sequence be located atleast 100-200 nucleotides from the AUG initiation codon and at least50-100 nucleotides away from the termination codon of the target mRNA(Duxbury (2004) J. Surgical Res. 117:339-344).

Researchers have determined that certain characteristics are common insiRNA molecules that effectively silence their target gene (Duxbury(2004) J. Surgical Res. 117:339-344; Ui-Tei et al. (2004) Nucl. AcidsRes. 32:936-48). As a general guide, siRNAs that include one or more ofthe following conditions are particularly useful in gene silencing inmammalian cells:GC ratio of between 45-55%, no runs of more than 9 G/Cresidues, G/C at the 5′ end of the sense strand; A/U at the 5′ end ofthe antisense strand; and at least 5 A/U residues in the first 7 basesof the 5′ terminal of the antisense strand.

siRNA are, in general, from about 10 to about 30 nucleotides in length.For example, the siRNA can be 10-30 nucleotides long, 12-28 nucleotideslong, 15-25 nucleotides long, 19-23 nucleotides long, or 21-23nucleotides long. When an siRNA contains two strands of differentlengths, the longer of the strands designates the length of the siRNA.In this situation, the unpaired nucleotides of the longer strand wouldform an overhang.

The term siRNA includes short hairpin RNAs (shRNAs). shRNAs comprise asingle strand of RNA that forms a stem-loop structure, where the stemconsists of the complementary sense and antisense strands that comprisea double-stranded siRNA, and the loop is a linker of varying size. Thestem structure of shRNAs generally is from about 10 to about 30nucleotides long. For example, the stem can be 10-30 nucleotides long,12-28 nucleotides long, 15-25 nucleotides long, 19-23 nucleotides long,or 21-23 nucleotides long.

Tools to assist siRNA design are readily available to the public. Forexample, a computer-based siRNA design tool is available on theinternet.

Synthesis of dsRNA and siRNA

dsRNA and siRNA can be synthesized chemically or enzymatically in vitroas described in Micura (2002) Agnes Chem. Int. Ed. Emgl. 41:2265-2269;Betz (2003) Promega Notes 85:15-18; and Paddison and Hannon (2002)Cancer Cell. 2:17-23. Chemical synthesis can be performed via manual orautomated methods, both of which are well known in the art as describedin Micura (2002), supra. siRNA can also be endogenously expressed insidethe cells in the form of shRNAs as described in Yu et al. (2002) Proc.Natl. Acad. Sci. USA 99:6047-6052; and McManus et al. (2002) RNA8:842-850. Endogenous expression has been achieved using plasmid-basedexpression systems using small nuclear RNA promoters, such as RNApolymerase III U6 or H1, or RNA polymerase II U1 as described inBrummelkamp et al. (2002) Science 296:550-553 (2002); and Novarino etal. (2004) J. Neurosci. 24:5322-5330.

In vitro enzymatic dsRNA and siRNA synthesis can be performed using anRNA polymerase mediated process to produce individual sense andantisense strands that are annealed in vitro prior to delivery into thecells of choice as describe in Fire et al. (1998) Nature 391:806-811;Donze and Picard (2002) Nucl. Acids Res. 30(10):e46; Yu et al. (2002);and Shim et al. (2002) J. Biol. Chem. 277:30413-30416. Severalmanufacturers (Promega, Ambion, New England Biolabs, and Stragene)produce transcription kits useful in performing the in vitro synthesis.

In vitro synthesis of siRNA can be achieved, for example, by using apair of short, duplex oligonucleotides that contain T7 RNA polymerasepromoters upstream of the sense and antisense RNA sequences as the DNAtemplate. Each oligonucleotide of the duplex is a separate template forthe synthesis of one strand of the siRNA. The separate short RNA strandsthat are synthesized are then annealed to form siRNA as described inProtocols and Applications, Chapter 2: RNA interference, PromegaCorporation, (2005).

In vitro synthesis of dsRNA can be achieved, for example, by using a T7RNA polymerase promoter at the 5′-ends of both DNA target sequencestrands. This is accomplished by using separate DNA templates, eachcontaining the target sequence in a different orientation relative tothe T7 promoter, transcribed in two separate reactions. The resultingtranscripts are mixed and annealed post-transcriptionally. DNA templatesused in this reaction can be created by PCR or by using two linearizedplasmid templates, each containing the T7 polymerase promoter at adifferent end of the target sequence. Protocols and Applications,Chapter 2: RNA interference, Promega Corporation, (2005).

In order to express the proteins described herein, delivery of nucleicacid sequences encoding the gene of interest can be delivered by severaltechniques. Examples of which include viral technologies (e.g.retroviral vectors, adenovirus vectors, adeno-associated virus vectors,alphavirus vectors and the like) and non-viral technologies (e.g.DNA/liposome complexes, micelles and targeted viral protein-DNAcomplexes) as described herein. Once inside the cell of interest,expression of the transgene can be under the control of ubiquitouspromoters (e.g. EF-1α) or tissue specific promoters (e.g. CalciumCalmodulin kinase 2 (CaMKI) promoter, NSE promoter and human Thy-1promoter). Alternatively expression levels may controlled by use of aninducible promoter system (e.g. Tet on/off promoter) as described inWiznerowicz et al. (2005) Stem Cells 77:8957-8961.

Non-limiting examples of promoters include, but are not limited to, thecytomegalovirus (CMV) promoter (Kaplitt et al. (1994) Nat. Genet.8:148-154), CMV/human β3-globin promoter (Mandel et al. (1998) J.Neurosci. 18:4271-4284), NCX1 promoter, αMHC promoter, MLC2v promoter,GFAP promoter (Xu et al. (2001) Gene Ther., 8:1323-1332), the 1.8-kbneuron-specific enolase (NSE) promoter (Klein et al. (1998) Exp. Neurol.150:183-194), chicken beta actin (CBA) promoter (Miyazaki (1989) Gene79:269-277) and the β-glucuronidase (GUSB) promoter (Shipley et al.(1991) Genetics 10:1009-1018), the human serum albumin promoter, thealpha-1-antitrypsin promoter. To improve expression, other regulatoryelements may additionally be operably linked to the transgene, such as,e.g., the Woodchuck Hepatitis Virus Post-Regulatory Element (WPRE)(Donello et al. (1998) J. Virol. 72: 5085-5092) or the bovine growthhormone (BGH) polyadenylation site.

Also provided herein is a polynucleotide probe or primer comprising atleast 10, or alternatively, at least 17 or alternatively at least 20, oralternatively, at least 50, or alternatively, at least 75polynucleotides, or alternatively at least 100 polynucleotides encodingSEQ ID NOS: 12 through 15 or their complements. Suitable probes andprimers are described supra. It is known in the art that a “perfectlymatched” probe is not needed for a specific hybridization. Minor changesin probe sequence achieved by substitution, deletion or insertion of asmall number of bases do not affect the hybridization specificity. Ingeneral, as much as 20% base-pair mismatch (when optimally aligned) canbe tolerated. A probe useful for detecting the aforementioned mRNA is atleast about 80% identical to the homologous region of comparable sizecontained in the previously identified sequences (identified above)which correspond to previously characterized polynucleotides of thisinvention. Alternatively, the probe is 85% identical to thecorresponding gene sequence after alignment of the homologous region;and yet further, it exhibits 90% identity, or still further, at least95% identical.

These probes can be used in radioassays (e.g. Southern and Northern blotanalysis) to detect or monitor expression of the polynucleotides orpolypeptides of this invention. The probes also can be attached to asolid support or an array such as a chip for use in high throughputscreening assays for the detection of expression of the genecorresponding to one or more polynucleotide(s) of this invention.

The polynucleotides and fragments of the polynucleotides of the presentinvention also can serve as primers for the detection of genes or genetranscripts that are expressed in neuronal cells, for example, toconfirm transduction of the polynucleotides into host cells. In thiscontext, amplification means any method employing a primer-dependentpolymerase capable of replicating a target sequence with reasonablefidelity. Amplification may be carried out by natural or recombinantDNA-polymerases such as T7 DNA polymerase, Klenow fragment of E. coliDNA polymerase, and reverse transcriptase. Primer length is the same asthat identified for probes, above.

The invention further provides the isolated polynucleotides of thisinvention operatively linked to a promoter of RNA transcription, as wellas other regulatory sequences for replication and/or transient or stableexpression of the DNA or RNA. As used herein, the term “operativelylinked” means positioned in such a manner that the promoter will directtranscription of RNA off the DNA molecule. Examples of such promotersare SP6, T4 and T7. In certain embodiments, cell-specific promoters areused for cell-specific expression of the inserted polynucleotide.Vectors which contain a promoter or a promoter/enhancer, withtermination codons and selectable marker sequences, as well as a cloningsite into which an inserted piece of DNA can be operatively linked tothat promoter are well known in the art and commercially available. Forgeneral methodology and cloning strategies, see Gene ExpressionTechnology (Goeddel ed., Academic Press, Inc. (1991)) and referencescited therein and Vectors: Essential Data Series (Gacesa and Ramji,eds., John Wiley & Sons, N.Y. (1994)), which contains maps, functionalproperties, commercial suppliers and a reference to GenEMBL accessionnumbers for various suitable vectors. Preferable, these vectors arecapable of transcribing RNA in vitro or in vivo.

Expression vectors containing these nucleic acids are useful to obtainhost vector systems to produce proteins and polypeptides. It is impliedthat these expression vectors must be replicable in the host organismseither as episomes or as an integral part of the chromosomal DNA.Suitable expression vectors include plasmids, viral vectors, includingadenoviruses, adeno-associated viruses, retroviruses, cosmids, etc.Adenoviral vectors are particularly useful for introducing genes intotissues in vivo because of their high levels of expression and efficienttransformation of cells both in vitro and in vivo. When a nucleic acidis inserted into a suitable host cell, e.g., a prokaryotic or aeukaryotic cell and the host cell replicates, the protein can berecombinantly produced. Suitable host cells will depend on the vectorand can include mammalian cells, animal cells, human cells, simiancells, insect cells, yeast cells, and bacterial cells as described aboveand constructed using well known methods. See Sambrook and Russell(2001), supra. In addition to the use of viral vector for insertion ofexogenous nucleic acid into cells, the nucleic acid can be inserted intothe host cell by methods well known in the art such as transformationfor bacterial cells; transfection using calcium phosphate precipitationfor mammalian cells; DEAE-dextran; electroporation; or microinjection.See Sambrook and Russell (2001), supra for this methodology.

The present invention also provides delivery vehicles suitable fordelivery of a polynucleotide of the invention into cells (whether invivo, ex vivo, or in vitro). A polynucleotide of the invention can becontained within a gene delivery vehicle, a cloning vector or anexpression vector. These vectors (especially expression vectors) can inturn be manipulated to assume any of a number of forms which may, forexample, facilitate delivery to and/or entry into a cell.

These isolated host cells containing the polynucleotides of thisinvention are useful for the recombinant replication of thepolynucleotides and for the recombinant production of peptides and forhigh throughput screening.

The polynucleotides of this invention can be conjugated to a detectablelabel or combined with a carrier such as a solid support orpharmaceutically acceptable carrier. Suitable solid supports aredescribed above as well as have suitable labels. Methods for attaching alabel to a polynucleotide are known to those skilled in the art. SeeSambrook and Russell (2001), supra.

Therapeutic Antibody Compositions

This invention also provides an antibody capable of specifically forminga complex with a protein or polypeptide of this invention, which areuseful in the therapeutic methods of this invention. The term “antibody”includes polyclonal antibodies and monoclonal antibodies, antibodyfragments, as well as derivatives thereof (described above). Theantibodies include, but are not limited to mouse, rat, and rabbit orhuman antibodies. Antibodies can be produced in cell culture, in phage,or in various animals, including but not limited to cows, rabbits,goats, mice, rats, hamsters, guinea pigs, sheep, dogs, cats, monkeys,chimpanzees, apes, etc. The antibodies are also useful to identify andpurify therapeutic polypeptides.

This invention also provides an antibody-peptide complex comprisingantibodies described above and a polypeptide that specifically binds tothe antibody. In one aspect the polypeptide is the polypeptide againstwhich the antibody was raised. In one aspect the antibody-peptidecomplex is an isolated complex. In a further aspect, the antibody of thecomplex is, but not limited to, a polyclonal antibody, a monoclonalantibody, a humanized antibody or an antibody derivative describedherein. Either or both of the antibody or peptide of theantibody-peptide complex can be detectably labeled. In one aspect, theantibody-peptide complex of the invention can be used as a control orreference sample in diagnostic or screening assays.

Polyclonal antibodies of the invention can be generated usingconventional techniques known in the art and are well-described in theliterature. Several methodologies exist for production of polyclonalantibodies. For example, polyclonal antibodies are typically produced byimmunization of a suitable mammal such as, but not limited to, chickens,goats, guinea pigs, hamsters, horses, mice, rats, and rabbits. Anantigen is injected into the mammal, which induces the B-lymphocytes toproduce IgG immunoglobulins specific for the antigen. This IgG ispurified from the mammals serum. Variations of this methodology includemodification of adjuvants, routes and site of administration, injectionvolumes per site and the number of sites per animal for optimalproduction and humane treatment of the animal. For example, adjuvantstypically are used to improve or enhance an immune response to antigens.Most adjuvants provide for an injection site antigen depot, which allowsfor a slow release of antigen into draining lymph nodes. Other adjuvantsinclude surfactants which promote concentration of protein antigenmolecules over a large surface area and immunostimulatory molecules.Non-limiting examples of adjuvants for polyclonal antibody generationinclude Freund's adjuvants, Ribi adjuvant system, and Titermax.Polyclonal antibodies can be generated using methods described in U.S.Pat. Nos. 7,279,559; 7,119,179; 7,060,800; 6,709,659; 6,656,746;6,322,788; 5,686,073; and 5,670,153.

The monoclonal antibodies of the invention can be generated usingconventional hybridoma techniques known in the art and well-described inthe literature. For example, a hybridoma is produced by fusing asuitable immortal cell line (e.g., a myeloma cell line such as, but notlimited to, Sp2/0, Sp2/0-AG14, NSO, NS1, NS2, AE-1, L.5, >243,P3X63Ag8.653, Sp2 SA3, Sp2 MAI, Sp2 SS1, Sp2 SA5, U397, MLA 144, ACT IV,MOLT4, DA-1, JURKAT, WEHI, K-562, COS, RAJI, NIH 3T3, HL-60, MLA 144,NAMAIWA, NEURO 2A, CHO, PerC.6, YB2/O) or the like, or heteromyelomas,fusion products thereof, or any cell or fusion cell derived therefrom,or any other suitable cell line as known in the art, with antibodyproducing cells, such as, but not limited to, isolated or cloned spleen,peripheral blood, lymph, tonsil, or other immune or B cell containingcells, or any other cells expressing heavy or light chain constant orvariable or framework or CDR sequences, either as endogenous orheterologous nucleic acid, as recombinant or endogenous, viral,bacterial, algal, prokaryotic, amphibian, insect, reptilian, fish,mammalian, rodent, equine, ovine, goat, sheep, primate, eukaryotic,genomic DNA, cDNA, rDNA, mitochondrial DNA or RNA, chloroplast DNA orRNA, hnRNA, mRNA, tRNA, single, double or triple stranded, hybridized,and the like or any combination thereof. Antibody producing cells canalso be obtained from the peripheral blood or, preferably the spleen orlymph nodes, of humans or other suitable animals that have beenimmunized with the antigen of interest. Any other suitable host cell canalso be used for expressing-heterologous or endogenous nucleic acidencoding an antibody, specified fragment or variant thereof, of thepresent invention. The fused cells (hybridomas) or recombinant cells canbe isolated using selective culture conditions or other suitable knownmethods, and cloned by limiting dilution or cell sorting, or other knownmethods.

In one embodiment, the antibodies described herein can be generatedusing a Multiple Antigenic Peptide (MAP) system. The MAP system utilizesa peptidyl core of three or seven radially branched lysine residues, onto which the antigen peptides of interest can be built using standardsolid-phase chemistry. The lysine core yields the MAP bearing about 4 to8 copies of the peptide epitope depending on the inner core thatgenerally accounts for less than 10% of total molecular weight. The MAPsystem does not require a carrier protein for conjugation. The highmolar ratio and dense packing of multiple copies of the antigenicepitope in a MAP has been shown to produce strong immunogenic response.This method is described in U.S. Pat. No. 5,229,490 and is hereinincorporated by reference in its entirety.

Other suitable methods of producing or isolating antibodies of therequisite specificity can be used, including, but not limited to,methods that select recombinant antibody from a peptide or proteinlibrary (e.g., but not limited to, a bacteriophage, ribosome,oligonucleotide, RNA, cDNA, or the like, display library; e.g., asavailable from various commercial vendors such as Cambridge AntibodyTechnologies (Cambridgeshire, UK), MorphoSys (Martinsrd/Planegg, Del.),Biovation (Aberdeen, Scotland, UK) BioInvent (Lund, Sweden), usingmethods known in the art. See U.S. Pat. Nos. 4,704,692; 5,723,323;5,763,192; 5,814,476; 5,817,483; 5,824,514; 5,976,862. Alternativemethods rely upon immunization of transgenic animals (e.g., SCID mice,Nguyen et al. (1977) Microbiol. Immunol. 41:901-907 (1997); Sandhu etal. (1996) Crit. Rev. Biotechnol. 16:95-118; Eren et al. (1998) Immunol.93:154-161 that are capable of producing a repertoire of humanantibodies, as known in the art and/or as described herein. Suchtechniques, include, but are not limited to, ribosome display (Hanes etal. (1997) Proc. Natl. Acad. Sci. USA, 94:4937-4942; Hanes et al. (1998)Proc. Natl. Acad. Sci. USA, 95:14130-14135); single cell antibodyproducing technologies (e.g., selected lymphocyte antibody method(“SLAM”) (U.S. Pat. No. 5,627,052, Wen et al. (1987) J. Immunol.17:887-892; Babcook et al., Proc. Natl. Acad. Sci. USA (1996)93:7843-7848); gel microdroplet and flow cytometry (Powell et al. (1990)Biotechnol. 8:333-337; One Cell Systems, (Cambridge, Mass.).; Gray etal. (1995) J. Imm. Meth. 182:155-163; and Kenny et al. (1995) Bio.Technol. 13:787-790); B-cell selection (Steenbakkers et al. (1994)Molec. Biol. Reports 19: 125-134.

Antibody derivatives of the present invention can also be prepared bydelivering a polynucleotide encoding an antibody of this invention to asuitable host such as to provide transgenic animals or mammals, such asgoats, cows, horses, sheep, and the like, that produce such antibodiesin their milk. These methods are known in the art and are described forexample in U.S. Pat. Nos. 5,827,690; 5,849,992; 4,873,316; 5,849,992;5,994,616; 5,565,362; and 5,304,489.

The term “antibody derivative” includes post-translational modificationto linear polypeptide sequence of the antibody or fragment. For example,U.S. Pat. No. 6,602,684 B1 describes a method for the generation ofmodified glycol-forms of antibodies, including whole antibody molecules,antibody fragments, or fusion proteins that include a region equivalentto the Fc region of an immunoglobulin, having enhanced Fc-mediatedcellular toxicity, and glycoproteins so generated.

Antibody derivatives also can be prepared by delivering a polynucleotideof this invention to provide transgenic plants and cultured plant cells(e.g., but not limited to tobacco, maize, and duckweed) that producesuch antibodies, specified portions or variants in the plant parts or incells cultured therefrom. For example, Cramer et al. (1999) Curr. Top.Microbol. Immunol. 240:95-118 and references cited therein, describe theproduction of transgenic tobacco leaves expressing large amounts ofrecombinant proteins, e.g., using an inducible promoter. Transgenicmaize have been used to express mammalian proteins at commercialproduction levels, with biological activities equivalent to thoseproduced in other recombinant systems or purified from natural sources.See, e.g., Hood et al. (1999) Adv. Exp. Med. Biol. 464:127-147 andreferences cited therein. Antibody derivatives have also been producedin large amounts from transgenic plant seeds including antibodyfragments, such as single chain antibodies (scFv's), including tobaccoseeds and potato tubers. See, e.g., Conrad et al. (1998) Plant Mol.Biol. 38:101-109 and reference cited therein. Thus, antibodies of thepresent invention can also be produced using transgenic plants,according to know methods.

Antibody derivatives also can be produced, for example, by addingexogenous sequences to modify immunogenicity or reduce, enhance ormodify binding, affinity, on-rate, off-rate, avidity, specificity,half-life, or any other suitable characteristic. Generally part or allof the non-human or human CDR sequences are maintained while thenon-human sequences of the variable and constant regions are replacedwith human or other amino acids.

In general, the CDR residues are directly and most substantiallyinvolved in influencing antigen binding. Humanization or engineering ofantibodies of the present invention can be performed using any knownmethod such as, but not limited to, those described in U.S. Pat. Nos.5,723,323; 5,976,862; 5,824,514; 5,817,483; 5,814,476; 5,763,192;5,723,323; 5,766,886; 5,714,352; 6,204,023; 6,180,370; 5,693,762;5,530,101; 5,585,089; 5,225,539; and 4,816,567.

Techniques for making partially to fully human antibodies are known inthe art and any such techniques can be used. According to oneembodiment, fully human antibody sequences are made in a transgenicmouse which has been engineered to express human heavy and light chainantibody genes. Multiple strains of such transgenic mice have been madewhich can produce different classes of antibodies. B cells fromtransgenic mice which are producing a desirable antibody can be fused tomake hybridoma cell lines for continuous production of the desiredantibody. (See for example, Russel et al. (2000) Infection and ImmunityApril 2000:1820-1826; Gallo et al. (2000) European J. of Immun.30:534-540; Green (1999) J. of Immun. Methods 231:11-23; Yang et al.(1999A) J. of Leukocyte Biology 66:401-410; Yang (1999B) Cancer Research59(6):1236-1243; Jakobovits. (1998) Advanced Drug Delivery Reviews31:33-42; Green and Jakobovits (1998) J. Exp. Med. 188(3):483-495;Jakobovits (1998) Exp. Opin. Invest. Drugs 7(4):607-614; Tsuda et al.(1997) Genomics 42:413-421; Sherman-Gold (1997) Genetic Engineering News17(14); Mendez et al. (1997) Nature Genetics 15:146-156; Jakobovits(1996) Weir's. Handbook of Experimental Immunology, The IntegratedImmune System Vol. IV, 194.1-194.7; Jakobovits (1995) Current Opinion inBiotechnology 6:561-566; Mendez et al. (1995) Genomics 26:294-307;Jakobovits (1994) Current Biology 4(8):761-763; Arbones et al. (1994)Immunity 1(4):247-260; Jakobovits (1993) Nature 362(6417):255-258;Jakobovits et al. (1993) Proc. Natl. Acad. Sci. USA 90(6):2551-2555; andU.S. Pat. No. 6,075,181.)

The antibodies of this invention also can be modified to create chimericantibodies. Chimeric antibodies are those in which the various domainsof the antibodies' heavy and light chains are coded for by DNA from morethan one species. See, e.g., U.S. Pat. No. 4,816,567.

Alternatively, the antibodies of this invention can also be modified tocreate veneered antibodies. Veneered antibodies are those in which theexterior amino acid residues of the antibody of one species arejudiciously replaced or “veneered” with those of a second species sothat the antibodies of the first species will not be immunogenic in thesecond species thereby reducing the immunogenicity of the antibody.Since the antigenicity of a protein is primarily dependent on the natureof its surface, the immunogenicity of an antibody could be reduced byreplacing the exposed residues which differ from those usually found inanother mammalian species antibodies. This judicious replacement ofexterior residues should have little, or no, effect on the interiordomains, or on the interdomain contacts. Thus, ligand binding propertiesshould be unaffected as a consequence of alterations which are limitedto the variable region framework residues. The process is referred to as“veneering” since only the outer surface or skin of the antibody isaltered, the supporting residues remain undisturbed.

The procedure for “veneering” makes use of the available sequence datafor human antibody variable domains compiled by Kabat et al. (1987)Sequences of Proteins of Immunological Interest, 4th ed., Bethesda, Md.,National Institutes of Health, updates to this database, and otheraccessible U.S. and foreign databases (both nucleic acid and protein).Non-limiting examples of the methods used to generate veneeredantibodies include EP 519596; U.S. Pat. No. 6,797,492; and described inPadlan et al. (1991) Mol. Immunol. 28(4-5):489-498.

The term “antibody derivative” also includes “diabodies” which are smallantibody fragments with two antigen-binding sites, wherein fragmentscomprise a heavy chain variable domain (VH) connected to a light chainvariable domain (VL) in the same polypeptide chain. (See for example, EP404,097; WO 93/11161; and Hollinger et al., (1993) Proc. Natl. Acad.Sci. USA 90:6444-6448.) By using a linker that is too short to allowpairing between the two domains on the same chain, the domains areforced to pair with the complementary domains of another chain andcreate two antigen-binding sites. (See also, U.S. Pat. No. 6,632,926 toChen et al. which discloses antibody variants that have one or moreamino acids inserted into a hypervariable region of the parent antibodyand a binding affinity for a target antigen which is at least about twofold stronger than the binding affinity of the parent antibody for theantigen.)

The term “antibody derivative” further includes “linear antibodies”. Theprocedure for making linear antibodies is known in the art and describedin Zapata et al. (1995) Protein Eng. 8(10):1057-1062. Briefly, theseantibodies comprise a pair of tandem Fd segments (V_(H)-C_(H)1-VH-C_(H)1) which form a pair of antigen binding regions. Linearantibodies can be bispecific or monospecific.

The antibodies of this invention can be recovered and purified fromrecombinant cell cultures by known methods including, but not limitedto, protein A purification, ammonium sulfate or ethanol precipitation,acid extraction, anion or cation exchange chromatography,phosphocellulose chromatography, hydrophobic interaction chromatography,affinity chromatography, hydroxylapatite chromatography and lectinchromatography. High performance liquid chromatography (“HPLC”) can alsobe used for purification.

Antibodies of the present invention include naturally purified products,products of chemical synthetic procedures, and products produced byrecombinant techniques from a eukaryotic host, including, for example,yeast, higher plant, insect and mammalian cells, or alternatively from aprokaryotic cells as described above.

If a monoclonal antibody being tested binds with protein or polypeptide,then the antibody being tested and the antibodies provided by thehybridomas of this invention are equivalent. It also is possible todetermine without undue experimentation, whether an antibody has thesame specificity as the monoclonal antibody of this invention bydetermining whether the antibody being tested prevents a monoclonalantibody of this invention from binding the protein or polypeptide withwhich the monoclonal antibody is normally reactive. If the antibodybeing tested competes with the monoclonal antibody of the invention asshown by a decrease in binding by the monoclonal antibody of thisinvention, then it is likely that the two antibodies bind to the same ora closely related epitope. Alternatively, one can pre-incubate themonoclonal antibody of this invention with a protein with which it isnormally reactive, and determine if the monoclonal antibody being testedis inhibited in its ability to bind the antigen. If the monoclonalantibody being tested is inhibited then, in all likelihood, it has thesame, or a closely related, epitopic specificity as the monoclonalantibody of this invention.

The term “antibody” also is intended to include antibodies of allisotypes. Particular isotypes of a monoclonal antibody can be preparedeither directly by selecting from the initial fusion, or preparedsecondarily, from a parental hybridoma secreting a monoclonal antibodyof different isotype by using the sib selection technique to isolateclass switch variants using the procedure described in Steplewski, etal. (1985) Proc. Natl. Acad. Sci. USA 82:8653 or Spira, et al. (1984) J.Immunol. Methods 74:307.

The isolation of other hybridomas secreting monoclonal antibodies withthe specificity of the monoclonal antibodies of the invention can alsobe accomplished by one of ordinary skill in the art by producinganti-idiotypic antibodies. Herlyn, et al. (1986) Science 232:100. Ananti-idiotypic antibody is an antibody which recognizes uniquedeterminants present on the monoclonal antibody produced by thehybridoma of interest.

Idiotypic identity between monoclonal antibodies of two hybridomasdemonstrates that the two monoclonal antibodies are the same withrespect to their recognition of the same epitopic determinant. Thus, byusing antibodies to the epitopic determinants on a monoclonal antibodyit is possible to identify other hybridomas expressing monoclonalantibodies of the same epitopic specificity.

It is also possible to use the anti-idiotype technology to producemonoclonal antibodies which mimic an epitope. For example, ananti-idiotypic monoclonal antibody made to a first monoclonal antibodywill have a binding domain in the hypervariable region which is themirror image of the epitope bound by the first monoclonal antibody.Thus, in this instance, the anti-idiotypic monoclonal antibody could beused for immunization for production of these antibodies.

In some aspects of this invention, it will be useful to detectably ortherapeutically label the antibody. Suitable labels are described supra.Methods for conjugating antibodies to these agents are known in the art.For the purpose of illustration only, antibodies can be labeled with adetectable moiety such as a radioactive atom, a chromophore, afluorophore, or the like. Such labeled antibodies can be used fordiagnostic techniques, either in vivo, or in an isolated test sample.

The coupling of antibodies to low molecular weight haptens can increasethe sensitivity of the antibody in an assay. The haptens can then bespecifically detected by means of a second reaction. For example, it iscommon to use haptens such as biotin, which reacts avidin, ordinitrophenol, pyridoxal, and fluorescein, which can react with specificanti-hapten antibodies. See, Harlow and Lane (1988) supra.

Antibodies can be labeled with a detectable moiety such as a radioactiveatom, a chromophore, a fluorophore, or the like. Such labeled antibodiescan be used for diagnostic techniques, either in vivo, or in an isolatedtest sample. Antibodies can also be conjugated, for example, to apharmaceutical agent, such as chemotherapeutic drug or a toxin. They canbe linked to a cytokine, to a ligand, to another antibody. Suitableagents for coupling to antibodies to achieve an antitumor effect includecytokines, such as interleukin 2 (IL-2) and Tumor Necrosis Factor (TNF);photosensitizers, for use in photodynamic therapy, including aluminum(III) phthalocyanine tetrasulfonate, hematoporphyrin, andphthalocyanine; radionuclides, such as iodine-131 (¹³¹I), yttrium-90(⁹⁰Y), bismuth-212 (²¹²Bi), bismuth-213 (²¹³Bi), technetium-99m(^(99m)Tc), rhenium-186 (¹⁸⁶Re), and rhenium-188 (¹⁸⁸Re); antibiotics,such as doxorubicin, adriamycin, daunorubicin, methotrexate, daunomycin,neocarzinostatin, and carboplatin; bacterial, plant, and other toxins,such as diphtheria toxin, pseudomonas exotoxin A, staphylococcalenterotoxin A, abrin-A toxin, ricin A (deglycosylated ricin A and nativericin A), TGF-alpha toxin, cytotoxin from Chinese cobra (naja najaatra), and gelonin (a plant toxin); ribosome inactivating proteins fromplants, bacteria and fungi, such as restrictocin (a ribosomeinactivating protein produced by Aspergillus restrictus), saporin (aribosome inactivating protein from Saponaria officinalis), and RNase;tyrosine kinase inhibitors; ly207702 (a difluorinated purinenucleoside); liposomes containing anti cystic agents (e.g., antisenseoligonucleotides, plasmids which encode for toxins, methotrexate, etc.);and other antibodies or antibody fragments, such as F(ab).

The antibodies of the invention also can be bound to many differentcarriers. Thus, this invention also provides compositions containing theantibodies and another substance, active or inert. Examples ofwell-known carriers include glass, polystyrene, polypropylene,polyethylene, dextran, nylon, amylases, natural and modified celluloses,polyacrylamides, agaroses and magnetite. The nature of the carrier canbe either soluble or insoluble for purposes of the invention. Thoseskilled in the art will know of other suitable carriers for bindingmonoclonal antibodies, or will be able to ascertain such, using routineexperimentation.

III. Methods of the Invention

One aspect of the present invention relates to a method of preventing orreducing bleeding in a subject undergoing anticoagulant therapy byadministering to the subject an effective amount of a factor Xa proteinderivative. In one embodiment, the derivative has a modified active siteand/or a modified Gla-domain thereby having either reduced or noprocoagulant activity. The derivative acts as an antidote andsubstantially neutralizes the anticoagulant activity of the inhibitor.In one embodiment, the derivative is either Gla-deficient orGla-domainless. The subject may be a mammal or more particularly, ahuman.

In another embodiment, the invention is directed to a method forselectively binding and inhibiting an exogenously administered factor Xainhibitor in a subject. The method comprises administering to thepatient an effective amount of a derivative of a factor Xa derivative asdescribed above. The subject may be a cell or a mammal, such as a human.

Patients suitable for this therapy have undergone prior anticoagulanttherapy, for example, they have been administered one or more of ananticoagulant, such as an inhibitor of factor Xa. Examples ofanticoagulants that are factor Xa inhibitors, include but are notlimited to, fondaparinux, idraparinux, biotinylated idraparinux,enoxaparin, fragmin, NAP-5, rNAPc2, tissue factor pathway inhibitor,DX-9065a, YM-60828, YM-150, apixaban, rivaroxaban, PD-348292,otamixaban, edoxaban, LY517717, GSK913893, low molecular weight heparin,and betrixaban, or any combination thereof. The source of variousanticoagulants is found throughout the description.

In one aspect, the derivative has a modified active site and/or amodified or removed Gla domain. In one aspect, the factor Xa derivativehas or exhibits no procoagulant activity. In this aspect, the derivativecomprises at least amino acid residues 40 to 448, 45 to 448, or 46 to448 of SEQ ID NO. 3 or equivalents thereof. In another aspect, thederivative comprises at least amino acid residues 45 to 139 and 195 to448 or 46 to 139 and 195-448 of SEQ ID NO. 3 or equivalents thereof.

In another aspect of the invention, the fXa derivative retains the threedimensional structure of the active site of the fXa protein. Informationregarding the three-dimensional structure of the des-Gla fXa may befound in Brandstetter, H et al. J. Bio. Chem., 1996, 271:29988-29992.

In another aspect of the invention, the fXa derivatives are completelylacking the light chain. Other modifications of the heavy chain maycomprise the catalytic domain of related serine proteases which arecapable of binding inhibitors. The related serine proteases havecatalytic domains which possess sufficient structural similarity to fXacatalytic domain and are therefore capable of binding small molecule fXainhibitors. Examples of related serine proteases include, but are notlimited to, mammalian proteases such as plasma kallikrein, thrombin andtrypsin or the bacterial protease subtilisin. These derivatives furtherinclude modifications at the amino acids residues equivalent to theactive site serine (SER379) or aspartic acid (ASP282) residues describedherein.

In some embodiments, the factor Xa protein with reduced procoagulantactivity comprises a modified light chain, wherein the modification issubstitution, addition or deletion of the Gla-domain to reduce thephospholipid membrane binding of fXa. In some embodiments, the primeamino acid sequence of fXa is not changed, but the side chain of certainamino acids has been changed. Examples of the modified Gla-domain thatreduces the phospholipid membrane binding of fXa comprises polypeptidesor proteins having the primary amino acid sequence of SEQ ID NO. 3 or anequivalent thereof, with at least one amino acid substitution, addition,or deletion as compared to the Gla-domain of a wild type human factor Xaprotein. In some embodiments, at least one amino acid being substitutedor deleted is a γ-carboxyglutamic acid (Gla). Gla residues are shown inSEQ ID NO. 3 at amino acid positions 6, 7, 14, 16, 19, 20, 25, 26, 29,32, and 39. In some embodiments, the antidote's primary amino acidsequence is identical to SEQ ID NO. 3 or equivalent thereof, but is anuncarboxylated, undercarboxylated or decarboxylated factor Xa protein.In some embodiments, the antidote is a des-Gla anhydro-fXa or des-GlafX-S379A. In some embodiments, the factor Xa protein with reducedphospholipid membrane binding further comprises modification or deletionof the EGF1 and/or EGF2 (shown in FIG. 3 as amino acids 46 to 84 and 85to 128, respectively) or a part, i.e. fragment of the EGF1 and/or EGF2domains. In some embodiments, the entire light chain or substantiallythe entire light chain is modified or removed. For example, the modifiedfXa protein with reduced phospholipid membrane binding may contain onlythe heavy chain or the modified fXa may contain the heavy chain and afragment of the light chain that contains Cys 132, the amino acidresidue that forms the single disulfide bond with Cys302 of the heavychain in SEQ ID NO. 3. In some embodiments, the derivative comprises theamino acid sequence of SEQ ID NO. 12. In some embodiments, thederivative is the two chain polypeptide comprising SEQ ID NO. 13. Inother embodiments, the derivative is the polypeptide of SEQ ID NO. 15.In other embodiments, the derivative is the polypeptide of SEQ ID NOs.19-25.

In some embodiments, the factor Xa protein derivative comprises amodified heavy chain that contains the catalytic domain of said factorXa protein. In some embodiments, at least one amino acid substitution ispresent at one or more amino acid position of fXa selected from thegroup consisting of Glu216, Glu218, Arg332, Arg347, Lys351, and Ser379in SEQ ID NOS. 3 and 7 (Glu37, Glu39, Arg150, Arg165, Lys169, and Ser195in chymotrypsin numbering, respectively). In some embodiments, theantidote is a factor Xa protein with active site serine (Ser379 in SEQID NOS. 3 and 7, Ser195 in chymotrypsin numbering) residue modified todehydro-alanine or alanine. Such modifications may be made to wild typefXa protein or to any of the modified fXa proteins or fragmentsdescribed above. For example, the des-Gla anhydro-fXa with active siteserine residues replaced by dehydro-alanine described in Example 1 hasshown antidote activity.

In other embodiments, the derivative has reduced interaction withcofactors fV/fVa and fVIII/fVIIIa as compared to wild-type or naturallyoccurring factor Xa. In some embodiments, at least one amino acidsubstitution is present at amino acid position Arg306, Glu310, Arg347,Lys351, Lys414 or Arg424 in SEQ ID NOS. 3 and 7 (Arg125, Glu 129,Arg165, Lys 169, Lys230 or Arg240 in chymotrypsin numbering,respectively). Such modifications may be made to wild type fXa proteinor to any of the modified fXa proteins or fragments described above.

In other embodiments, the antidote is a protein comprising the aminoacid sequence of a serine protease catalytic domain which can mimic theinhibitor binding capability of the fXa heavy chain. Such proteins mayinclude mammalian proteases such as plasma kallikrein, thrombin, trypsin(or its bacterial homolog subtilisin) which have been recombinantlymodified to lack serine protease activity capable of cleaving proteinsubstrates but still possess the structural characteristics of theactive site cleft.

Also provided by this invention are pharmaceutical compositionscontaining one or more of the modified factor Xa derivatives and apharmaceutically acceptable carrier. The compositions are administeredto a subject in need thereof in an amount that will provide the desiredbenefit, a reduction or stopping of bleeding. The compositions can beco-administered with any suitable agent or therapy that complements orenhances the activity of the factor Xa derivative. An example of such isa second agent capable of extending the plasma half-life of theantidote. Examples of suitable second agents include but are not limitedto an anti-fXa antibody recognizing the exosite of fXa heavy chain or analpha-2-macroglobulin bound fXa derivative. Formation of the complexbetween fXa derivative and a second agent (exosite antibody oralpha-2-macroglobulin) would block macromolecular interactions butretains the ability of active site dependent inhibitor bindings.Examples of anti-fXa antibodies suitable for co-administration includebut are not limited to those described in Yang Y. H., et al, J. Immunol.2006, 1; 177(11):8219-25, Wilkens, M and Krishnaswamy, S., J. Bio.Chem., 2002, 277 (11), 9366-9374, and Church W R, et al, Blood, 1988,72(6), 1911-1921.

In some embodiments, a factor Xa protein is modified by chemical,enzymatic or recombinant means. For example, the active site Ser379 maybe chemically modified to dehydroalanine, and the Gla domain may beenzymatically removed by chymotrypsin digestion as described inExample 1. A modified fXa described herein may also be produced byrecombinant means by modifying the sequence of the cDNA encodingwild-type fX (SEQ ID NO. 2) described in more details in Example 7 fordirect expression of recombinant antidote (r-Antidote) or alternatively,a fX protein with the desired modification may be produced byrecombinant means followed by activation to the modified fXa by anactivator, such as a snake venom, e.g. Russell's viper venom, andcomplexes of fVIIa/tissue factor or fIXa/fVIIIa.

Subjects that will benefit from the administration of the compositionsdescribed herein and the accompanying methods include those that areexperiencing, or predisposed to a clinical major bleeding event or aclinically significant non-major bleeding event. Examples of clinicalmajor bleeding events are selected from the group consisting ofhemorrhage, bleeding into vital organs, bleeding requiring re-operationor a new therapeutic procedure, and a bleeding index of ≧2.0 with anassociated overt bleed. (Turpie A G G, et al, NEJM, 2001, 344: 619-625.)Additionally, the subject may be experiencing or predisposed to anon-major bleeding event selected from the group consisting of epistaxisthat is persistent or recurrent and in substantial amount or will notstop without intervention, rectal or urinary tract bleeding that doesnot rise to a level requiring a therapeutic procedure, substantialhematomas at injection sites or elsewhere that are spontaneous or occurwith trivial trauma, substantial blood loss more than usually associatedwith a surgical procedure that does not require drainage, and bleedingrequiring unplanned transfusion.

In some embodiments, the antidote is administered after theadministration of an overdose of a fXa inhibitor or prior to a surgery,which may expose subjects to the risk of hemorrhage.

In any of the methods described herein, it should be understood, even ifnot always explicitly stated, that an effective amount of the derivativeis administered to the subject. The amount can be empirically determinedby the treating physician and will vary with the age, gender, weight andhealth of the subject. Additional factors to be considered by thetreating physician include but are not limited to the identity and/oramount of factor Xa inhibitor, which may have been administered, themethod or mode that the antidote will be administered to the subject,the formulation of the antidote, and the therapeutic end point for thepatient. With these variables in mind, one of skill will administer atherapeutically effective amount to the subject to be treated. It iscontemplated that a therapeutically effective amount of the antidotesdescribed herein sufficient to counteract, or substantially neutralize,an anticoagulant in a subject may contain from about 0.01 milligram ofantidote per kilogram of a subject's body weight to 1 gram of antidoteper kilogram of a subject's body weight of antidote. It is furthercontemplated that the antidote may be provided to the subject in aconcentration a range of from about 10 nanomolar to about 100micromolar, or about 10 nanomolar to about 5 micromolar, or about 100nanomolar to about 2.5 micromolar.

The compositions can be administered in amounts that are effective forthe antidote to selectively recognize and bind, either directly orindirectly, the factor Xa inhibitor in the subject. They also can beadministered in amounts to substantially inhibit or substantiallyneutralize exogenously administered factor Xa inhibitors in a subject.

In still another aspect, the invention relates to a pharmaceuticalcomposition for reversing or neutralizing the anticoagulant activity ofa factor Xa inhibitor administered to a subject, comprisingadministering an effective amount of an antidote to the factor Xainhibitor and a pharmaceutically acceptable carrier, with the provisothat the antidote is not plasma derived factor VIIa, recombinant factorVIIa, fresh frozen plasma, prothrombin complex concentrates and wholeblood.

In some embodiments, the antidote is any one of the antidotes asdescribed above. In some embodiments, the antidote is conjugated with amoiety capable of extending the circulating half-life of the antidote.In some embodiments, the moiety is selected from the group consisting ofpolyethylene glycol, an acyl group, a liposome, a carrier protein, anartificial phospholipid membrane, and a nanoparticle. For example, anon-active site lysine or cysteine residue of a fXa derivative describedherein may be chemically modified to attach to a polyethylene glycolmolecule. Other methods provided in Werle, M. & Bernkop-Schnürch, A.Strategies to Improve Plasma Half Life Time of Peptide and ProteinDrugs, Amino Acids 2006, 30(4):351-367 may be used to extend the plasmahalf life of the antidotes of this invention.

In other embodiments of the invention, the half-life of the fXaderivative is improved by coupling the antidote to Fc carrier domains.In one embodiment, the antidote is coupled to an Fc fragment, such as animmunoglobulin peptide portion or an IgG1 fragment. In one embodiment, achimeric protein is contemplated which comprises the fXa derivative andthe immunoglobulin peptide portion. In yet another embodiment, the fXaderivative and the immunoglobulin peptide is coupled by a chemicalreaction, such as a disulfide bond with the human IgG heavy chain andkappa light chain constant regions.

In some embodiments, the pharmaceutical composition further comprises anagent capable of extending the plasma half-life of the antidote. Inanother aspect, the pharmaceutical composition has been co-formulatedwith an agent capable of extending the plasma half-life of the antidote.In some embodiments, the co-administered or co-formulated agent is ananti-fXa antibody recognizing the exosite of fXa or analpha-2-macroglobulin bound fXa derivative.

IV. Therapies

The present invention relates to a therapeutic method of preventing orreducing bleeding in a subject undergoing anticoagulant therapy. It iscontemplated that the antidotes or derivatives of the present inventionmay be short-duration drugs to be used in elective or emergencysituations which can safely and specifically neutralize a fXainhibitor's conventional anticoagulant properties without causingdeleterious hemodynamic side-effects or exacerbation of theproliferative vascular response to injury.

In one embodiment, the therapeutically effective amount of an antidoteexhibits a high therapeutic index. The therapeutic index is the doseratio between toxic and therapeutic effects which can be expressed asthe ratio between LD₅₀ and ED₅₀. The LD₅₀ is the dose lethal to 50% ofthe population and the ED₅₀ is the dose therapeutically effective in 50%of the population. The LD₅₀ and ED₅₀ are determined by standardpharmaceutical procedures in animal cell cultures or experimentalanimals. The antidotes or derivatives of this invention may beadministered once or several times when needed to neutralize the effectof a fXa inhibitor present in a subject's plasma. Preferably, theantidotes of this invention is sufficient when administered in a singledose.

It is contemplated that a typical dosage of the antidotes of theinvention will depend on the actual clinical setting and inhibitorconcentration in plasma. In in vitro assay, such as thrombin generation,clinical clotting assays such as aPTT, PT and ACT, a therapeuticallyeffective amount of an antidote is expected to produce a correction ofex vivo clotting activity of 10% or more. In vitro assays indicate thatan antidote/inhibitor ratio >1.0 should show reversal effect. Themaximum plasma concentration for antidote is expected to be in the micromolar range, probably at 10 micromolar or below.

In a clinical setting, one of the criteria in determining theeffectiveness of an antidote is that it produces any change of actualmeasures of bleeding. In clinical trials, categories of major bleedsinclude fatal hemorrhage, bleeds into vital organs (intracranial,intraocular, retroperitoneal, spinal, pericardial), any bleed requiringre-operation or a new therapeutic procedure (e.g., aspiration of anoperated knee, thoracotomy tube insertion for hemothorax, endoscopicelectrocoagulation, etc) or a bleeding index of ≧2.0 if it is associatedwith an overt bleed. The bleeding index is defined as the number ofunits of packed red cells or whole blood transfused plus the hemoglobinvalues before the bleeding episode minus the hemoglobin values after thebleed has stabilized (in grams per deciliter).

Another criterion for antidote efficacy in clinical settings is that itreduces clinically significant non-major bleeding. This category ofhemorrhages include bleeding that is not major but is more than usualand warrants clinical attention, including epistaxis that is persistentor recurrent and in substantial amount or will not stop withoutintervention; rectal or urinary tract bleeding that does not rise to alevel requiring a therapeutic procedure (e.g., new insertion of a Foleycatheter or cystoscopic inspection), substantial hematomas at injectionsites or elsewhere that are spontaneous or occur with trivial trauma;substantial blood loss; bleeding requiring unplanned transfusion. Asused herein, “substantial blood loss” refers to amount of blood lossthat is more than that amount usually associated with surgicalprocedure. Substantial blood loss leads to swelling that is managedconservatively because it falls short of requiring drainage.

In one embodiment, the derivatives of this invention have sufficientplasma circulating half life for substantially neutralizing the fXainhibitor present in plasma. Activated fXa has essentially nocirculating half life in humans, as it is effectively inhibited byATIII, TFPI and other plasma inhibitors (Fuchs, H. E. and Pizzo, S. V.,J. Clin. Invest., 1983, 72:2041-2049). Inactive fXa has been shown tohave a circulating half-life of 2-3 hours in humans. In a baboon model,the half-life of a fXa blocked in the active site by DEGR([5-(dimethylamino)1-naphthalenesulfonyl]-glutamylglycylarginylchloromethyl ketone) was approximately 10 hours or 2 hours, asdetermined by isotopic or enzyme-linked immunosorbent assays,respectively (Taylor, F. B. et al, Blood, 1991, 78(2):364-368).

It may be desirable to extend the half life of an antidote fXaderivative to 24-48 hours. It is contemplated that conjugation oraddition of one or more of the following moieties will increase theplasma half life of an antidote:

a) polyethylene glycol;

b) an acyl group;

c) liposomes and encapsulating agents;

d) carrier proteins;

e) artificial phospholipid membrane;

f) immunoglobulin; and

g) nanoparticle.

The conjugation site may not be limited to special chain or residue solong as the conjugation does not mask the inhibitor binding site(s) ofthe antidote. The antidotes described herein may be administered incombination with any one or more than one of the compounds describedabove.

In general, administered antibodies have much longer half life thancirculating blood coagulation proteins. It is possible to use a complexconsisting of Gla-domain deficient fXa and an antibody bound to theexosite of fXa as an antidote with extended circulating half life.Formation of a complex between fXa and the antibody targeting theexosite may reduce interaction of an Gla-domain deficient fXa withmacromolecular substrates and inhibitors, such as prothrombin andantithrombin III, while leaving the active site cleft unperturbed sothat the complex can act as an antidote to bind active site directedsmall molecule inhibitor. Formation of α-2-macroglobulin-fXa complex canalso be of useful as an antidote for fXa small molecule inhibitors.

Efficacy of the antidotes in reversal of the anticoagulant activity offXa inhibitors as well as its procoagulant activity may be determined byin vitro assays and animal models by those of skill in the art. Examplesof in vitro assays are thrombin generation, anti-fXa units, clinicalclotting assays such as aPTT, PT and ACT. An antidote of this inventionis contemplated to be capable of producing 10% or more correction of exvivo clotting activity. Several in vivo animal models of bleeding timeand/or blood loss in, for example, rodents, such as mice, dogs andprimates, such as monkeys, may be used to measure efficacy.

V. Pharmaceutical Compositions

The present invention further provides compositions comprising a fXaderivative and a pharmaceutically acceptable carrier.

“Pharmaceutically acceptable carriers” refers to any diluents,excipients, or carriers that may be used in the compositions of theinvention. Pharmaceutically acceptable carriers include ion exchangers,alumina, aluminum stearate, lecithin, serum proteins, such as humanserum albumin, buffer substances, such as phosphates, glycine, sorbicacid, potassium sorbate, partial glyceride mixtures of saturatedvegetable fatty acids, water, salts or electrolytes, such as protaminesulfate, disodium hydrogen phosphate, potassium hydrogen phosphate,sodium chloride, zinc salts, colloidal silica, magnesium trisilicate,polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol,sodium carboxymethylcellulose, polyacrylates, waxes,polyethylene-polyoxypropylene-block polymers, polyethylene glycol andwool fat. Suitable pharmaceutical carriers are described in Remington'sPharmaceutical Sciences, Mack Publishing Company, a standard referencetext in this field. They are preferably selected with respect to theintended form of administration, that is, oral tablets, capsules,elixirs, syrups and the like, and consistent with conventionalpharmaceutical practices.

The pharmaceutical compositions of the invention can be manufactured bymethods well known in the art such as conventional granulating, mixing,dissolving, encapsulating, lyophilizing, or emulsifying processes, amongothers. Compositions may be produced in various forms, includinggranules, precipitates, or particulates, powders, including freezedried, rotary dried or spray dried powders, amorphous powders,injections, emulsions, elixirs, suspensions or solutions. Formulationsmay optionally contain stabilizers, pH modifiers, surfactants,bioavailability modifiers and combinations of these.

Pharmaceutical formulations may be prepared as liquid suspensions orsolutions using a sterile liquid, such as oil, water, alcohol, andcombinations thereof. Pharmaceutically suitable surfactants, suspendingagents or emulsifying agents, may be added for oral or parenteraladministration. Suspensions may include oils, such as peanut oil, sesameoil, cottonseed oil, corn oil and olive oil. Suspension preparation mayalso contain esters of fatty acids, such as ethyl oleate, isopropylmyristate, fatty acid glycerides and acetylated fatty acid glycerides.Suspension formulations may include alcohols, such as ethanol, isopropylalcohol, hexadecyl alcohol, glycerol and propylene glycol. Ethers, suchas poly(ethyleneglycol), petroleum hydrocarbons, such as mineral oil andpetrolatum, and water may also be used in suspension formulations.

The compositions of this invention are formulated for pharmaceuticaladministration to a mammal, preferably a human being. Suchpharmaceutical compositions of the invention may be administered in avariety of ways, preferably parenterally.

It is contemplated that in order to quickly reverse the anticoagulantactivity of a fXa inhibitor present in a patient's plasma in a emergencysituation, the antidote of this invention can or may be administered tothe systemic circulation via parental administration. The term“parenteral” as used herein includes subcutaneous, intravenous,intramuscular, intra-articular, intra-synovial, intrasternal,intrathecal, intrahepatic, intralesional and intracranial injection orinfusion techniques. However, in cases where the fXa inhibitor beingneutralized has a long plasma half life, a continuous infusion or asustained release formulation may be required to bind to the fXainhibitor and such free up the active fXa prior to the clearance of thefXa inhibitor from the body.

Sterile injectable forms of the compositions of this invention may beaqueous or oleaginous suspension. These suspensions may be formulatedaccording to techniques known in the art using suitable dispersing orwetting agents and suspending agents. The sterile injectable preparationmay also be a sterile injectable solution or suspension in a non-toxicparenterally acceptable diluent or solvent, for example as a solution in1,3-butanediol. Among the acceptable vehicles and solvents that may beemployed are water, Ringer's solution and isotonic sodium chloridesolution. In addition, sterile, fixed oils are conventionally employedas a solvent or suspending medium. For this purpose, any bland fixed oilmay be employed including synthetic mono- or di-glycerides. Fatty acids,such as oleic acid and its glyceride derivatives are useful in thepreparation of injectables, as are natural pharmaceutically-acceptableoils, such as olive oil or castor oil, especially in theirpolyoxyethylated versions. These oil solutions or suspensions may alsocontain a long-chain alcohol diluent or dispersant, such ascarboxymethyl cellulose or similar dispersing agents which are commonlyused in the formulation of pharmaceutically acceptable dosage formsincluding emulsions and suspensions. Other commonly used surfactants,such as Tweens, Spans and other emulsifying agents or bioavailabilityenhancers which are commonly used in the manufacture of pharmaceuticallyacceptable solid, liquid, or other dosage forms may also be used for thepurposes of formulation. Compounds may be formulated for parenteraladministration by injection such as by bolus injection or continuousinfusion. A unit dosage form for injection may be in ampbules or inmulti-dose containers.

In addition to dosage forms described above, pharmaceutically acceptableexcipients and carriers and dosage forms are generally known to thoseskilled in the art and are included in the invention. It should beunderstood that a specific dosage and treatment regimen for anyparticular patient will depend upon a variety of factors, including theactivity of the specific antidote employed, the age, body weight,general health, sex and diet, renal and hepatic function of the patient,and the time of administration, rate of excretion, drug combination,judgment of the treating physician or veterinarian and severity of theparticular disease being treated.

VI. Kits

The invention further provides kits or packages. In some embodiments,the kit of the present invention comprises: (a) a first containercontaining a fXa inhibitor for regular administration for the treatmentof thrombosis, and (b) a second container containing an antidote of thisinvention to be used in cases when there is an overdose of the fXainhibitor in (a) or when normal hemostasis needs to be restored to stopor prevent bleeding. In other embodiments, the kit further comprises alabel explaining when these two agents in (a) and (b) should be used.

The first and second container can be a bottle, jar, vial, flask,syringe, tube, bag, or any other container used in the manufacture,storage, or distribution of a pharmaceutical product. The package insertcan be a label, tag, marker, or the like, that recites informationrelating to the pharmaceutical composition of the kit. The informationrecited will usually be determined by the regulatory agency governingthe area in which the pharmaceutical composition is to be sold, such asthe United States Food and Drug Administration. Preferably, the packageinsert specifically recites the indications for which the pharmaceuticalcomposition has been approved. The package insert may be made of anymaterial on which a person can read information contained therein orthereon. Preferably, the package insert is a printable material, such aspaper, adhesive-backed paper cardboard, foil, or plastic, and the like,on which the desired information has been printed or applied.

EXAMPLES

The invention is further understood by reference to the followingexamples, which are intended to be purely exemplary of the invention.The present invention is not limited in scope by the exemplifiedembodiments, which are intended as illustrations of single aspects ofthe invention only. Any methods that are functionally equivalent arewithin the scope of the invention. Various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and accompanyingfigures. Such modifications fall within the scope of the appendedclaims.

Unless otherwise stated all temperatures are in degrees Celsius. Also,in these examples and elsewhere, abbreviations have the followingmeanings:

aa=amino acid

ab=antibody

ACT=activated clotting time

aPTT=activated partial thromboplastin time

CHO cell=Chinese hamster ovary cell

CHO dhfr(−) cells=CHO cells lacking dhfr gene

fXa=factor Xa

hr=hour

INR=international normalized ratio

IV=intravenous

kg=kilogram

M=molar

mg=milligram

mg/kg=milligram/kilogram

mg/mL=milligram/milliliter

min=minute

mL=milliliter

mM=millimolar

nm=nanometer

nM=nanomolar

PO=oral

PRP=platelet rich plasma

PT=prothrombin time

RFU=relative fluorescence unit

s=second

TF=tissue factor

U/mL=units/milliliter

μL or uL=microliter

μM=micromolar

μg=microgram

Example 1 Preparation of Des-Gla Anhydro-FXa by Chymotrypsin Digestion

Des-Gla anhydro-fXa was prepared according to the procedure of Morita,T. et al., J. Bio. Chem., 1986, 261(9):4015-4023 by incubatinganhydro-fXa, in which dehydroalanine replaces the active-site serine,with chymotrypsin in 0.05 M Tris-HCl, 0.1 M NaCl, at pH 7.5 and 22° C.for 60 minutes. In a typical experiment setting, 0.5milligrams/milliliter (mg/mL) anhydro-fXa was incubated with 5units/milliliter (U/mL) chymotrypsin-agarose beads with gentleagitation. At the end of the reaction, the α-chymotrypsin-agarose beadswere removed by centrifugation or filtration. This was followed byincubation with excess amount of inhibitors4-amidino-phenyl-methane-sulfonyl fluoride (APMSF), tosyl-L-lysinechloromethyl ketone (TLCK), and tosyl-L-phenylalanine chloromethylketone (TPCK) to quench the residual fXa activity or any activity ofchymotrypsin possibly leached from the beads. Gla-domain fragment andinhibitors were removed from the final product, des-Gla anhydro-fXa, byan Amicon Ultra. Centrifugal filter device (YM10 membrane) or byconventional dialysis. Concentrating or buffer exchange, if necessary,was also achieved at the same time.

The Gla-containing anhydro-fXa was prepared according to the procedurereported by Nogami et al., J. Biol. Chem., 1999, 274(43):31000-7. Asshown in FIG. 5, the Gla-containing anhydro-fXa has diminished enzymaticactivity but is capable of binding fXa inhibitors such as betrixaban.This is described in detail in Example 4.

Chymotrypsin digestion of active fXa can be carried out according toabove procedure without using APMSF. Clotting activity of active fXa wasdetermined before the chymotrypsin digestion, and after 15, 30 and 60minutes of chymotrypsin digestion according to the procedure describedin Example 3 below. FIG. 7 shows complete loss of clotting activityafter 30 minutes of chymotrypsin digestion. The incubation time wereextended to 60 minutes to ensure complete removal of the Gla domain.

Example 2 Thrombin Generation Assay in Platelet Poor Plasma (PPP) orPlatelet Rich Plasma (PRP)

In this example, human platelet poor or platelet rich plasma sampleswere prepared from blood of healthy donors drawn into 0.32% citrate. PRPand PPP were prepared by spinning the anticoagulated blood at ˜100×gravity or 1000× gravity for 20 minutes, respectively, at roomtemperature. 75-100 microliter (uL) plasma was mixed with CaCl₂ andZ-Gly-Gly-Arg-aminomethylcoumarin (Z-GGR-AMC, a thrombin fluorogenicsubstrate). Tissue factor (Innovin, Dade Behring) was added to initiatethe generation of thrombin. For a typical experiment, the reactionmixture contained 15 millimolar (mM) Ca²⁺, 100 micromolar (μM)Z-GGR-AMC, and 0.1 nanomolar (nM) tissue factor (TF) (Innovin). Thrombinformation was monitored continuously at 37° C. by a fluorometric platereader (Molecular Devices) measuring the relative fluorescence units(RFU). Inhibitor and antidote, when present, were pre-incubated withplasma for 20 minutes at room temperature before initiation of thrombingeneration.

The results of various experiments using this assay may be found inFIGS. 4, 6, and 9.

Example 3 Clotting Prolongation Assays

Two clotting assay formats were used to test the effects of factor Xainhibitors and the antidote on clotting prolongation. In the firstformat, a 96-well plate was used to measure multiple samples at the sametime. In the second assay format, aPTT was measured with a conventionalcoagulation instrument (MLA Electra 800 automatic coagulation timer).

In the 96-well plate format method, human platelet poor plasma orplatelet rich plasma was prepared similarly as procedures in Example 2.75-100 μL plasma was recalcified with CaCl₂, incubated at 37° C. for 3minutes and clot formation was initiated by adding tissue factor(Innovin, Dade Behring) or an aPTT reagent (Actin FS, Dade Behring).Change of OD405 was monitored continuously by a plate reader (MolecularDevices). Clotting time was defined as the time (second) when the halfmaximal value of absorbance (OD405 nm) change was reached. Factor Xainhibitor and antidote, when present, were pre-incubated with plasma atroom temperature for 20 minutes before initiation of the reaction.

When an active fXa was tested for its clotting activity as shown in FIG.7, 75-100 uL fX deficient plasma (George King Bio-Medical, Inc.) wasrecalcified with CaCl₂, incubated at 37° C. for 3 minutes and fXaproducts following chymotrypsin digestion was added to the plasma toinitiate clot formation. Change of OD405 was continuously monitored by aplate reader as described before.

In FIG. 13, the effect of 400 nM betrixaban on aPTT prolongation ofnormal human plasma and the reversal of betrixaban inhibitory effect byantidote des-Gla anhydro-fXa was measured with a MLA Electra 800Automatic coagulation timer. 100 μL pooled human plasma was mixed with400 nM betrixaban and different concentration of antidote. aPTT reagent(Actin FS, Dade Behring) and CaCl₂ were added per manufacturer'sinstructions for measurement of clotting times.

Results of additional experiments using this assay may be found in FIGS.10 and 11.

Example 4 Reversal of Inhibition of fXa by Betrixaban by Anhydro-fXa ordes-Gla Anhydro-fXa

To measure the inhibition of fXa activity by betrixaban and reversal ofits inhibitory effect, purified active fXa, different concentrations ofbetrixaban and anhydro-fXa or des-Gla anhydro-fXa were added to 20 mMTris, 150 mM NaCl, 5 mM Ca²⁺, and 0.1% Bovine Serum Albumin (BSA). Afterincubation at room temperature for 20 minutes, 100 μM Spectrozyme-fXa (afactor Xa chromogenic substrate, American Diagnostica) was added to themixture and the rate of substrate cleavage was monitored continuouslyfor 5 minutes at 405 nanometer (nm) by a plate reader. In FIG. 5, thechromogenic activity was normalized to active fXa in the absence of anyinhibitor. Initial velocity of product formation as a function ofinhibitor and antidote concentration was analyzed by nonlinearregression to estimate the affinity of betrixaban to the antidote (FIG.8).

The effect of the antidote des-Gla anhydro-fXa on thrombin activitytoward a chromogenic substrate S2288 (200 μM) was measured similarly asbefore with or without Argatroban, a specific small molecule IIainhibitor. As expected, the antidote (538 nM) does not affect theamidolytic activity of IIa (5 nM) or its inhibition by 50 nM Argatroban.

Example 5 Preparation of fXa with Decarboxylated γ-Carboxyglutamic AcidResidues

A fXa derivative with decarboxylated γ-carboxyglutamic acid residues canbe prepared by treating fXa protein, for example, based on the procedurereported by Bajaj, et al. J. Biol. Chem., 1982, 257(7):3726-3731. 2 to 5mg of purified or recombinant fXa in 2 mL of 0.1 Molar ammoniumbicarbonate at pH 8.0 is lyophilized. The resulting powder is sealedunder a vacuum of less than 20 μm and heated at 110° C. for variousperiods of time to obtain decarboxylated fXa.

Example 6 Preparation of Recombinant des-Gla fXa-S379A

The fXa derivatives may be produced by recombinant DNA method with oneof the following procedures based on fX cDNA (SEQ ID NO. 2) forexpressing fX (SEQ ID NOS. 1, 3) or fXa derivatives (SEQ ID NOS. 4, 5,9, and 11) in a suitable host organism according to general proceduresof mutagenesis and molecular biology.

Recombinant fX and fX derivatives can be expressed in, for example,human embryonic kidney cells HEK293 based on procedures described inLarson, P. J., et al, Biochem., 1998, 37:5029-5038, and Camire, R. M.,et al, Biochem., 2000, 39, 14322-14329. Recombinant fX can be activatedto rfXa by factor X activator Russell's Viper Venom (RVV). rfXa can befurther processed to des-Gla anhydro-fXa based on procedures describedin Example 1.

Recombinant fX-S379A (S195A in chymotrypsin numbering) with the activesite serine residue being replaced by alanine, and preferably theactivated fXa mutant, rfXa-S379A, may be expressed, for example, inChinese Hamster Ovary (CHO) cells based on procedures described by Sinhaet al., Protein Expression and Purif. 1992, 3: 518-524; Wolf, D. L. etal, J. Biol. Chem., 1991, 266(21):13726-13730.

Des-Gla fXa-S379A may be prepared by chymotrypsin digestion of fXa-S379Aaccording to procedures described in Example 1.

More preferably, Des-Gla fXa-S379A may be expressed directly accordingto previous procedures with deletion of Gla-domain fragment bymutagenesis procedures. For example, recombinant protein expression canbe used to express: des-Gla(1-39)-fXa-S379A, after removal of Gla-domainfragment 1-39 of SEQ ID NO. 3; des-Gla(1-44)-fXa-S379A, equivalent toSEQ ID NO. 10 with dehydro-alanine being replaced by alanine; anddes-Gla(1-45)-fXa-S379A with entire Gla-domain being removed (SEQ ID NO.11).

Further truncations at EGF1 or EGF1 plus EGF2 domain (FIG. 2) can alsobe made to express des(1-84)-fXa-S379A or des(1-128)-fXa-S379Aderivatives.

Example 7 Expression of Recombinant fXa Mutant in CHO Cell

This example describes the recombinant protein expression construct andthe cell line for the direct expression of a Gla-domainless fXa-5379A(S195A in chymotrypsin numbering) variant. The recombinant antidote doesnot require activation or chemical modification steps necessary toproduce the pd-Antidote and has comparable affinity to the plasmaderived protein in the in vitro assays discussed herein.

In this example, a fXa mutant (SEQ ID NO. 13, Table 13) was directlyexpressed in CHO cell (see FIG. 14 for expression vector) and functionalprotein was purified from conditioned medium as described below.Recombinant antidote (r-Antidote) functional activity was tested invitro and in animal model (Example 8).

PCR was used to mutate the cDNA sequence of fX (SEQ ID NO. 2) in threeregions. The first mutation was the deletion of 6-39 aa in theGla-domain of FX (SEQ ID NO. 3, FIG. 3). The second mutation wasreplacing the activation peptide sequence 143-194 aa with -RKR- (SEQ IDNO: 40). This produced a -RKRRKR- linker (SEQ ID NO: 41) connecting thelight chain and the heavy chain. Upon secretion, this linker is removedin CHO resulting in a two-chain fXa molecule. The third mutation ismutation of active site residue S379 to an Ala residue.

The polypeptide produced by the cDNA (SEQ ID NO. 16) just described isdescribed in Table 12 (SEQ ID NO. 12). The alignment of the cDNA to thepolypeptide is shown in Table 17. The two-chain fXa molecule producedafter secretion is a light chain fragment described in Table 14 (SEQ IDNO. 14) and a heavy chain fragment described in Table 15 (SEQ ID NO.15).

The first 1-5 aa in fX sequence was reserved and used to connect thepolypeptide of fXa mutant to the prepro peptide of a (SEQ ID NO. 1, FIG.1), ensuring proper processing of the prepro peptide in fXa mutant.

DNA sequence encoding the polypeptide of fXa mutant described above wassequenced and inserted to the expression vector shown in FIG. 14. Thepolynucleotide of the expression vector is shown in SEQ ID NO. 17.Plasmid DNA was linearized and transfected into CHO dhfr(−) cells. Cellswere selected using tetrahydrofolate (HT) deficient media plusmethotrexate (MTX). Stable clones were screened for high proteinexpression using a fX ELISA kit (Enzyme Research Laboratories, CatalogueNumber FX-EIA). FXa mutant protein was expressed in serum free mediumand conditioned medium was harvested and processed for purification.

Target protein in the conditioned medium can be isolated by ion exchangechromatography and subsequently purified by single step affinitychromatography (such as an anti-fXa antibody coupled to a matrix) or bya combination of several chromatography steps such as hydrophobic andsize exclusion matrices. The affinity purifications may includechromatographic material that selectively binds to fXa active sitecleft, such as benzamidine-sepharose or soybean trypsininhibitor-agarose (STI-Agarose).

FIG. 15 shows the Western blots of affinity (STI-Agarose, Sigma Catalog#T0637) purified fXa mutant using monoclonal antibodies (Enzyme ResearchLaboratories, FX-EIA) recognizing fX heavy and light chain,respectively. Upon reduction of the disulfide bond which connects thelight and heavy chain, r-Antidote shows the heavy chain band of expectedmobility (similar to plasma derived fXa) in the Western blot. Deletionof amino acid residues 6-39 in the Gla-domain of fXa mutant results in alower molecular weight band for the light chain of r-Antidote comparedto plasma derived fXa. Position of the molecular weight marks can alsobe seen on the blot.

Figures B and 15C shows a SDS-PAGE and Western blot of purifiedr-Antidote by ion exchange and affinity purification followed by sizeexclusion chromatrography using a Superdex 75 10/300 GL column (GEHealthcare, Cat#17-5174-01).

Example 8 In Vivo Mouse Model

The pharmacokinetic and pharmacodynamic (PK-PD) profile of betrixaban inmale C57B1/6 mice with or without administrating antidote were tested.Single oral administration of betrixaban was dosed at 0, 15, 25, and 75mg/kg for controls groups. 15 mg/kg was used for antidote treated group.A single intravenous (IV) injection of antidote (300 ug/200 μL) orvehicle (normal saline, 200 μL) was administered 5 minutes prior to the1.5 hr. time point.

At 1.5, 2.0, and 4.0 hrs following oral administration of betrixaban,mice were anesthetized with a ketamine cocktail (SC) and exsanguinatedvia cardiac puncture. Blood samples (0.5 mL) were obtained in 50 μLtrisodium citrate. Whole blood INR was measured using Hemochron Jr.cartridges (International Technidyne Corporation) per the manufacturer'sinstructions. Mouse platelet poor plasma was prepared by centrifugationfor betrixaban and antidote (ELISA) plasma concentration determinations.

For recombinant antidote (r-Antidote) experiment, mice were orally dosedwith betrixaban at 0, 15, 25, and 75 mg/kg for control groups. 15 mg/kgwas used for antidote (300 μg/200 μL) treated group. Samples were takenat 1.5 hr after oral administration of betrixaban (5 min. followingantidote injection).

As shown in FIGS. 16 and 17 and Tables A and B, single injection (300μg, IV) of plasma derived antidote (pd-Antidote) or recombinant fXamutant (r-Antidote) to mice following administration of betrixaban (15mg/kg, PO) effectively captured the inhibitor in vivo. PK-PD correlationof whole blood INR and antidote plasma concentration (Tables A and B)indicated >50% reduction of functional betrixaban based on INRmeasurements, and justified effective neutralization of fXa inhibitorsby the antidote via multiple injections or other regimes. It iscontemplated that these results demonstrate that the fXa derivatives ofthis invention have potential of acting as universal antidotes toreverse the anticoagulant effect of fXa inhibitors in patients withbleeding or other medical emergencies.

TABLE A PK-PD correlation in pd-Antidote treated mice at 1.5 hr after 15mg/kg Betrixaban oral administration (5 min after antidote injection)pd-Antidote treated animal 1 2 3 4 5 6 7 Mean Betrixaban (ng/mL) 673 7931170 415 217 664 879 687 Expected INR 4.2 4.5 5.2 3.3 2.3 4.1 4.7 4.0Measured INR 2.3 2.3 3.3 0.8 0.8 1.5 2.0 1.9 % Correction 63.9 66.6 52.3100 100 83.1 74.4 77.2

TABLE B PK-PD correlation in r-Antidote treated mice at 1.5 hr after 15mg/kg Betrixaban oral administration (5 min after antidote injection)r-Antidote treated animal 1 2 3 4 Mean Betrixaban (ng/mL) 434 262 335494 381 Expected INR 3.2 2.5 2.8 3.5 3.0 Measured INR 2.0 0.9 1.2 0.91.3 % Correction 50.0 94.1 80.0 93.6 77.3

FIG. 22 shows mouse experiment with a single IV injection (1 injection)or two injections (2 injections) of the r-antidote (n=5 per group, 312ug/200 ul r-Antidote) following oral administration of betrixaban (15mg/kg). For the single injection group, mouse blood samples were takenat 1 hr. following oral administration of betrixaban. Vehicle(control_(—)1) or r-Antidote (1 injection) was administered 5 min priorto the 1 hr. time point. For the double injection group, vehicle orr-Antidote was injected at 55 min and repeated at 115 min following oraladministration of betrixaban. Mouse blood samples were taken at 2 hr.for vehicle (control_(—)2) and r-Antidote (2 injections) treated mice.Measured INR as a function of antidote/betrixaban ratio in mouse plasmafollowing single or double injections of the antidote was shown in FIG.22 B.

Example 9 In Vitro Reversal of Rivaroxaban and Apixaban by Antidote

As expected, the antidotes contemplated by this invention were also ableto bind and neutralize other active site directed fXa inhibitors. TablesC and D show in vitro correction of inhibition by betrixaban,rivaroxaban and apixaban by pd-Antidote and r-Antidote. Purified fXa(3.0 nM), inhibitor (7.5 nM), and different concentrations of antidotewere incubated for 10 min at 22° C. in a buffer with 20 mM Tris, 150 mMNaCl, 0.1% BSA, pH7.4. fXa activity was assayed similar to Example 4.

TABLE C % Correction of inhibition by fXa inhibitors pd-Antidote (nM)Betrixaban Rivaroxaban Apixaban 0 0 0 0 10.2 13.1 10.6 6.5 20.4 34.837.4 11.4 40.7 47.1 46.8 15.0 61.1 68.4 55.7 40.3 101.8 67.5 69.4 52.3162.9 80.5 74.0 56.0 203.7 82.6 72.6 60.2

TABLE D % Correction of inhibition by fXa inhibitors r-Antidote (nM)Betrixaban Rivaroxaban Apixaban 0 0 0 0 9.3 21.5 23.2 13.3 18.6 52.754.2 33.5 37.2 75.5 72.6 49.9 55.8 86.5 79.9 59.2 93.1 94.9 89.1 64.4148.9 99.3 96.7 74.8 186.1 99.5 94.8 72.6

As shown in Table C, 204 nM pd-Antidote produces at least 60% correctionof the inhibitory effects of tested inhibitors, while in Table D >95%correction of inhibition was achieved by the r-Antidote (186 nM) forbetrixaban and rivaroxaban, and >70% reversal of apixaban.

Example 10 In Vitro Reversal of Betrixaban by r-Antidote

In Table E, the effect of recombinant antidote protein on reversal ofanticoagulation by betrixaban was tested in a human plasma clottingassay. The effect of 300 nM and 400 nM betrixaban on aPTT prolongationof plasma and the reversal of inhibitory effect was measured by a MLAElectra 800 Automatic coagulation timer. 100 μL pooled citrateanticoagulated human plasma was mixed with 300 nM or 400 nM betrixabanand different concentrations of antidote. aPTT reagent (Actin FS, DadeBehring) and CaCl₂ were added per manufacturer's instructions.

TABLE E r-Antidote reversal of anticoagulant activity of betrixaban %Correction aPTT Fold of anti- (sec) Change coagulation Control humanplasma 35.2 1.00 — 300 nM Betrixaban 61.8 1.76 — 300 nM Betrixaban + 570nM r-Antidote 38.3 1.09 88 300 nM Betrixaban + 760 nM r-Antidote 38.21.09 88 300 nM Betrixaban + 1140 nM r-Antidote 38.1 1.08 90 400 nMBetrixaban 66.3 1.88 — 400 nM Betrixaban + 380 nM rAntidote 47.1 1.34 61400 nM Betrixaban + 570 nM rAntidote 39.9 1.13 85 400 nM Betrixaban +760 nM rAntidote 39.9 1.13 85 400 nM Betrixaban + 1140 nM rAntidote 37.81.07 92 400 nM Betrixaban + 1520 nM rAntidote 39.4 1.12 86 1140 nMrAntidote 38.9 1.11 — 1520 nM rAntidote 38.8 1.10 —

Example 11 In Vitro Reversal of Low Molecular Weight Heparin (“LMWH”) byr-Antidote

In FIG. 18, the effect of r-Antidote to reverse the inhibitory effect ofLMWH enoxaparin (Sanofi-Aventis) was tested by turbidity changes inhuman plasma. Enoxaparin (0-1.25 U/mL) was incubated at 22° C. for 20min with or without 508 nM r-Antidote. Turbidity changes were measuredaccording to procedures described in Example 3. 508 nM r-Antidotesubstantially corrected (>75%) the inhibitory effect of 0.3125-1.25 U/mLEnoxaparin.

In FIG. 19, the effect of r-Antidote on reversal of anticoagulation by alow molecular weight heparin (LMWH enoxaparin, Sanofi-Aventis) wastested in a human plasma clotting assay. The effect of 1 antiXa Unit/mLLMWH on aPTT prolongation of plasma and the reversal of inhibitoryeffect was measured by a MLA Electra 800 Automatic coagulation timer.100 μL pooled citrate anticoagulated human plasma was mixed withenoxaparin and different concentrations of antidote. Prior tomeasurement of clotting time, aPTT reagent (Actin FS, Dade Behring) andCaCl₂ were added per manufacturer's instructions. Addition of 1.14 μMrecombinant antidote produced a 52% correction of anticoagulationproduced by 1 Unit/mL enoxaparin.

Example 12 In Vitro Reversal of Rivaroxaban by r-Antidote

In FIG. 20, the effect of recombinant antidote protein on reversal ofanticoagulation by a small molecule factor Xa inhibitor (rivaroxaban,Bay 59-7939) was tested in a human plasma clotting assay. As reported byPerzborn et al, J. Thromb. Haemost. 3:514-521, 2005; prothrombin timemeasurements are an accurate method for evaluating the anticoagulanteffect of rivaroxaban. The effect of 1 μM rivaroxaban on prothrombintime (PT) prolongation of pooled human plasma and the reversal ofinhibitory effect was measured by a MLA Electra 800 Automaticcoagulation timer. 100 μL pooled citrate anticoagulated human plasma wasmixed with rivaroxaban and different concentrations of antidote. Priorto measurement of clotting time, rabbit brain Thromboplastin C Plusreagent (Dade Behring) was added to plasma samples per manufacturer'sinstructions. Addition of 1.9 μM recombinant antidote produced a 100%correction of anticoagulation produced by 1 μM rivaroxaban.

Example 13 In Vitro Reversal of Apixaban by r-Antidote

In Table F, the effect of recombinant antidote protein on reversal ofanticoagulation by apixaban was tested in a human plasma clotting assay.As reported by Pinto et al., J. Med. Chem. 55(22):5339-5356, 2007;prothrombin time (PT) measurements are an accurate method of evaluatingthe ex vivo anticoagulant effects of apixaban. The effect of 1 μM and1.5 μM apixaban on prothrombin time (PT) prolongation of pooled humanplasma and the reversal of inhibitory effect was measured by a MLAElectra 800 Automatic coagulation timer. 100 μL pooled citrateanticoagulated human plasma was mixed with apixaban and differentconcentrations of antidote. Prior to measurement of clotting time,rabbit brain Thromboplastin C Plus reagent (Dade Behring) was added toplasma samples per manufacturer's instructions. Addition of 1.9 μMrecombinant antidote produced a 97% correction of anticoagulationproduced by 1.5 μM apixaban.

TABLE F r-Antidote reversal of anticoagulant activity of Apixaban FoldPT (sec) Change Control human plasma 14.1 — 1 μM apixaban 16.4 1.16 1 μMapixaban + 380 nM rAntidote 15.3 1.09 1 μM apixaban + 760 nM rAntidote14.9 1.06 1 μM apixaban + 1.14 μM rAntidote 14.2 1.01 1 μM apixaban +1.52 μM rAntidote 14.2 1.01 1.5 μM apixaban 18.4 1.31 1.5 μM apixaban +1.52 μM rAntidote 14.6 1.04 1.5 μM apixaban + 1.90 μM rAntidote 14.31.01 1.52 μM rAntidote 14 — 1.90 μM rAntidote 14.2 —

Example 14 In Vitro Inhibition of Argatroban by des-Gla Anhydro-fXa

To measure the inhibition of thrombin activity by argatroban andreversal of its inhibitory effect, purified human thrombin (5 nM),argatroban (50 nM) and different concentrations of antidote des-Glaanhydro fXa were added to a buffer containing 20 mM Tris, 0.15 M NaCl, 5mM Calcium chloride, 0.1% bovine serum albumin, pH 7.4. After incubationat room temperature for 20 min, an amidolytic substrate 52288 (200 uM)was added to the mixture and the rate of p-nitroanilide substratecleavage was monitored by absorbance at 405 nm. The results arepresented in FIG. 12.

Example 15 Methods for Extending Plasma Half Life of Circulation ofAntidote Upon Human Dosing

Recent pharmacokinetic data in animals have shown that recombinantantidote expressed in CHO DuxB11 cells have a plasma half life ofclearance (T_(1/2)) of 19 minutes in SD rats and 30 minutes in rhesusmonkeys. A longer half life of clearance may be desirable for human use.Extension of T_(1/2) in humans may require modification of the proteinby a conjugated moiety capable of extending circulating half life of theaa derivative. The conjugated moiety such as an antibody may includewhole antibodies and any antigen binding fragment or a single chainthereof. Thus the term “antibody” includes any protein or peptidecontaining molecule that comprises at least a portion of animmunoglobulin molecule. Examples of such include, but are not limitedto a complementarity determining region (CDR) of a heavy or light chainor a ligand binding portion thereof, a heavy chain or light chainvariable region, a heavy chain or light chain constant region, aframework (FR) region, or any portion thereof, or at least one portionof a binding protein. The antibodies can be polyclonal or monoclonal andcan be isolated from any suitable biological source, e.g., murine, rat,sheep and canine. A conjugated moiety may also be selected from a groupconsisting of polyethylene glycol, an acyl group, a liposome, anencapsulating agent, a carrier protein, an artificial phospholipidmembrane, a nanoparticle, and combinations thereof.

Pharmacokinetics of Antidote in Rat

One milligram of antidote was administered as a short intravenousinfusion over five minutes to four Sprague-Dawley rats. Serial plasmasamples were collected and analyzed for antidote concentration using anenzyme-linked immunosorbent assay (ELISA). Plasma concentration-timeprofiles of the antidote can be described by a two compartment model.Systemic clearance of the antidote was low (1.65 mL/min/kg) and volumeof distribution was small (0.27 L/kg). The distribution half-life was 19minutes followed by a much longer terminal half-life of 10 hours.Because of the need to immediately maintain antidote plasmaconcentration above that of a factor Xa inhibitor (the anticoagulant),the distribution half-life is expected to have a much bigger impact thanthe terminal half-life on the dose selection during an overdosetreatment. The data is presented in FIG. 23.

Pharmacokinetics of Antidote in Rhesus Monkey

Two animals were each dosed with 10 mg antidote by intravenous dosingover a ten minute period. Plasma samples for determination of T_(1/2)were carried out in a manner similar to that in the rat study. Plasmahalf life of clearance (T_(1/2)) was approximately 30 minutes. The datais presented in FIG. 24.

It is to be understood that while the invention has been described inconjunction with the above embodiments, that the foregoing descriptionand examples are intended to illustrate and not limit the scope of theinvention. Other aspects, advantages and modifications within the scopeof the invention will be apparent to those skilled in the art to whichthe invention pertains.

TABLE 1 Sequence ID NO. 1 - Polypeptide Sequence of Human Factor X   1MGRPLHLVLL SASLAGLLLL GESLFIRREQ ANNILARVTR ANSFLEEMKK GHLERECMEE  61TCSYEEAREV FEDSDKTNEF WNKYKDGDQC ETSPCQNQGK CKDGLGEYTC TCLEGFEGKN 121CELFTRKLCS LDNGDCDQFC HEEQNSVVCS CARGYTLADN GKACIPTGPY PCGKQTLERR 181KRSVAQATSS SGEAPDSITW KPYDAADLDP TENPFDLLDF NQTQPERGDN NLTRIVGGQE 241CKDGECPWQA LLINEENEGF CGGTILSEFY ILTAAHCLYQ AKRFKVRVGD RNTEQEEGGE 301AVHEVEVVIK HNRFTKETYD FDIAVLRLKT PITFRMNVAP ACLPERDWAE STLMTQKTGI 361VSGFGRTHEK GRQSTRLKML EVPYVDRNSC KLSSSFIITQ NMFCAGYDTK QEDACQGDSG 421GPHVTRFKDT YFVTGIVSWG EGCARKGKYG IYTKVTAFLK WIDRSMKTRG LPKAKSHAPE 481VITSSPLK

TABLE 2 Sequence ID NO. 2 - A polynucleotide Sequence Encoding Factor X   1 gactttgctc cagcagcctg tcccagtgag gacagggaca cagtactcgg ccacaccatg  61 gggcgcccac tgcacctcgt cctgctcagt gcctccctgg ctggcctcct gctgctcggg 121 gaaagtctgt tcatccgcag ggagcaggcc aacaacatcc tggcgagggt cacgagggcc 181 aattcctttc ttgaagagat gaagaaagga cacctcgaaa gagagtgcat ggaagagacc 241 tgctcatacg aagaggcccg cgaggtcttt gaggacagcg acaagacgaa tgaattctgg 301 aataaataca aagatggcga ccagtgtgag accagtcctt gccagaacca gggcaaatgt 361 aaagacggcc tcggggaata cacctgcacc tgtttagaag gattcgaagg caaaaactgt 421 gaattattca cacggaagct ctgcagcctg gacaacgggg actgtgacca gttctgccac 481 gaggaacaga actctgtggt gtgctcctgc gcccgcgggt acaccctggc tgacaacggc 541 aaggcctgca ttcccacagg gccctacccc tgtgggaaac agaccctgga acgcaggaag 601 aggtcagtgg cccaggccac cagcagcagc ggggaggccc ctgacagcat cacatggaag 661 ccatatgatg cagccgacct ggaccccacc gagaacccct tcgacctgct tgacttcaac 721 cagacgcagc ctgagagggg cgacaacaac ctcaccagga tcgtgggagg ccaggaatgc 781 aaggacgggg agtgtccctg gcaggccctg ctcatcaatg aggaaaacga gggtttctgt 841 ggtggaacca ttctgagcga gttctacatc ctaacggcag cccactgtct ctaccaagcc 901 aagagattca aggtgagggt aggggaccgg aacacggagc aggaggaggg cggtgaggcg 961 gtgcacgagg tggaggtggt catcaagcac aaccggttca caaaggagac ctatgacttc1021 gacatcgccg tgctccggct caagaccccc atcaccttcc gcatgaacgt ggcgcctgcc1081 tgcctccccg agcgtgactg ggccgagtcc acgctgatga cgcagaagac ggggattgtg1141 agcggcttcg ggcgcaccca cgagaagggc cggcagtcca ccaggctcaa gatgctggag1201 gtgccctacg tggaccgcaa cagctgcaag ctgtccagca gcttcatcat cacccagaac1261 atgttctgtg ccggctacga caccaagcag gaggatgcct gccaggggga cagcgggggc1321 ccgcacgtca cccgcttcaa ggacacctac ttcgtgacag gcatcgtcag ctggggagag1381 ggctgtgccc gtaaggggaa gtacgggatc tacaccaagg tcaccgcctt cctcaagtgg1441 atcgacaggt ccatgaaaac caggggcttg cccaaggcca agagccatgc cccggaggtc1501 ataacgtcct ctccattaaa gtgagatccc actcaaaaaa aaaaaaaaaa aaaaaaaaaa

TABLE 3Sequence ID NO. 3 - Polypeptide Sequence of Mature Human Factor X   1ANSFLEEMKK GHLERECMEE TCSYEEAREV FEDSDKTNEF WNKYKDGDQC ETSPCQNQGK  61CKDGLGEYTC TCLEGFEGKN CELFTRKLCS LDNGDCDQFC HEEQNSVVCS CARGYTLADN 121GKACIPTGPY PCGKQTLERR KRSVAQATSS SGEAPDSITW KPYDAADLDP TENPFDLLDF 181NQTQPERGDN NLTRIVGGQE CKDGECPWQA LLINEENEGF CGGTILSEFY ILTAAHCLYQ 241AKRFKVRVGD RNTEQEEGGE AVHEVEVVIK HNRFTKETYD FDIAVLRLKT PITFRMNVAP 301ACLPERDWAE STLMTQKTGI VSGFGRTHEK GRQSTRLKML EVPYVDRNSC KLSSSFIITQ 361NMFCAGYDTK QEDACQGDSG GPHVTRFKDT YFVTGIVSWG EGCARKGKYG IYTKVTAFLK 421WIDRSMKTRG LPKAKSHAPE VITSSPLK

TABLE 4Sequence ID NO. 4 - Polypeptide Sequence of the Gla-domainless Factor Xalacking 1 to 44 amino acid residues Light Chain   1                                                KDGDQC ETSPCQNQGK  61CKDGLGEYTC TCLEGFEGKN CELFTRKLCS LDNGDCDQFC HEEQNSVVCS CARGYTLADN 121GKACIPTGPY PCGKQTLER Heavy Chain 181               IVGGQE CKDGECPWQA LLINEENEGF CGGTILSEFY ILTAAHCLYQ 241AKRFKVRVGD RNTEQEEGGE AVHEVEVVIK HNRFTKETYD FDIAVLRLKT PITFRMNVAP 301ACLPERDWAE STLMTQKTGI VSGFGRTHEK GRQSTRLKML EVPYVDRNSC KLSSSFIITQ 361NMFCAGYDTK QEDACQGDSG GPHVTRFKDT YFVTGIVSWG EGCARKGKYG IYTKVTAFLK 421WIDRSMKTRG LPKAKSHAPE VITSSPLK

TABLE 5Sequence ID NO. 5 - Polypeptide Sequence of the Gla-domainless Factor Xalacking 1 to 45 amino acid residues Light Chain   1                                                 DGDQC ETSPCQNQGK  61CKDGLGEYTC TCLEGFEGKN CELFTRKLCS LDNGDCDQFC HEEQNSVVCS CARGYTLADN 121GKACIPTGPY PCGKQTLER Heavy Chain 181               IVGGQE CKDGECPWQA LLINEENEGF CGGTILSEFY ILTAAHCLYQ 241AKRFKVRVGD RNTEQEEGGE AVHEVEVVIK HNRFTKETYD FDIAVLRLKT PITFRMNVAP 301ACLPERDWAE STLMTQKTGI VSGFGRTHEK GRQSTRLKML EVPYVDRNSC KLSSSFIITQ 361NMFCAGYDTK QEDACQGDSG GPHVTRFKDT YFVTGIVSWG EGCARKGKYG IYTKVTAFLK 421WIDRSMKTRG LPKAKSHAPE VITSSPLK

TABLE 6Sequence ID NO. 6 - Polypeptide Sequence of Activated Human Factor Xaprior to Post-Translation of Glutamic Acid to γ-Carboxyglutamic acidLight Chain   1ANSFLEEMKK GHLERECMEE TCSYEEAREV FEDSDKTNEF WNKYKDGDQC ETSPCQNQGK  61CKDGLGEYTC TCLEGFEGKN CELFTRKLCS LDNGDCDQFC HEEQNSVVCS CARGYTLADN 121GKACIPTGPY PCGKQTLER Heavy Chain 181               IVGGQE CKDGECPWQA LLINEENEGF CGGTILSEFY ILTAAHCLYQ 241AKRFKVRVGD RNTEQEEGGE AVHEVEVVIK HNRFTKETYD FDIAVLRLKT PITFRMNVAP 301ACLPERDWAE STLMTQKTGI VSGFGRTHEK GRQSTRLKML EVPYVDRNSC KLSSSFIITQ 361NMFCAGYDTK QEDACQGDSG GPHVTRFKDT YFVTGIVSWG EGCARKGKYG IYTKVTAFLK 421WIDRSMKTRG LPKAKSHAPE VITSSPLK

TABLE 7Sequence ID NO. 7 - Polypeptide Sequence of Activated Human Factor Xawith Post-Translation of Glutamic Acid to γ-Carboxyglutamic acid (γrepresents γ-Carboxyglutamic Acid Residue) Light Chain   1ANSFLγγMKK GHLγRγCMγγ TCSYγγARγV FγDSDKTNγF WNKYKDGDQC ETSPCQNQGK  61CKDGLGEYTC TCLEGFEGKN CELFTRKLCS LDNGDCDQFC HEEQNSVVCS CARGYTLADN 121GKACIPTGPY PCGKQTLER Heavy Chain 181               IVGGQE CKDGECPWQA LLINEENEGF CGGTILSEFY ILTAAHCLYQ 241AKRFKVRVGD RNTEQEEGGE AVHEVEVVIK HNRFTKETYD FDIAVLRLKT PITFRMNVAP 301ACLPERDWAE STLMTQKTGI VSGFGRTHEK GRQSTRLKML EVPYVDRNSC KLSSSFIITQ 361NMFCAGYDTK QEDACQGDSG GPHVTRFKDT YFVTGIVSWG EGCARKGKYG IYTKVTAFLK 421WIDRSMKTRG LPKAKSHAPE VITSSPLK

TABLE 8Sequence ID NO. 8 - Polypeptide Sequence of Activated Human Factor Xa-Light Chain with Post-Translation of Glutamic Acid to γ-Carboxyglutamicacid Light Chain   1 ANSFLγγMKK GHLγRγCMγγTCSYγγARγV FγDSDKTNγF WNKYKDGDQC ETSPCQNQGK  61CKDGLGEYTC TCLEGFEGKN CELFTRKLCS LDNGDCDQFC HEEQNSVVCS CARGYTLADN 121GKACIPTGPY PCGKQTLER

TABLE 9Sequence ID NO. 9 - Polypeptide Sequence of Activated Human Factor Xa-Heavy Chain 181               IVGGQE CKDGECPWQA LLINEENEGF CGGTILSEFY ILTAAHCLYQ 241AKRFKVRVGD RNTEQEEGGE AVHEVEVVIK HNRFTKETYD FDIAVLRLKT PITFRMNVAP 301ACLPERDWAE STLMTQKTGI VSGFGRTHEK GRQSTRLKML EVPYVDRNSC KLSSSFIITQ 361NMFCAGYDTK QEDACQGDSG GPHVTRFKDT YFVTGIVSWG EGCARKGKYG IYTKVTAFLK 421WIDRSMKTRG LPKAKSHAPE VITSSPLK

TABLE 10Sequence ID NO. 10 - Polypeptide Sequence of the Des-Gla Anhydro Factor Xa(Ã represents dehydroalanine) Light Chain   1                                                KDGDQC ETSPCQNQGK  61CKDGLGEYTC TCLEGFEGKN CELFTRKLCS LDNGDCDQFC HEEQNSVVCS CARGYTLADN 121GKACIPTGPY PCGKQTLER Heavy Chain 181               IVGGQE CKDGECPWQA LLINEENEGF CGGTILSEFY ILTAAHCLYQ 241AKRFKVRVGD RNTEQEEGGE AVHEVEVVIK HNRFTKETYD FDIAVLRLKT PITFRMNVAP 301ACLPERDWAE STLMTQKTGI VSGFGRTHEK GRQSTRLKML EVPYVDRNSC KLSSSFIITQ 361NMFCAGYDTK QEDACQGDÃG GPHVTRFKDT YFVTGIVSWG EGCARKGKYG IYTKVTAFLK 421WIDRSMKTRG LPKAKSHAPE VITSSPLK

TABLE 11Sequence ID NO. 11 - Polypeptide Sequence of the Des-Gla fXa-S379ALight Chain   1                                                 DGDQC ETSPCQNQGK  61CKDGLGEYTC TCLEGFEGKN CELFTRKLCS LDNGDCDQFC HEEQNSVVCS CARGYTLADN 121GKACIPTGPY PCGKQTLER Heavy Chain 181               IVGGQE CKDGECPWQA LLINEENEGF CGGTILSEFY ILTAAHCLYQ 241AKRFKVRVGD RNTEQEEGGE AVHEVEVVIK HNRFTKETYD FDIAVLRLKT PITFRMNVAP 301ACLPERDWAE STLMTQKTGI VSGFGRTHEK GRQSTRLKML EVPYVDRNSC KLSSSFIITQ 361NMFCAGYDTK QEDACQGDAG GPHVTRFKDT YFVTGIVSWG EGCARKGKYG IYTKVTAFLK 421WIDRSMKTRG LPKAKSHAPE VITSSPLK

TABLE 12Sequence ID NO. 12 - Polypeptide Sequence of a Human Factor Xa triplemutant prior to removal of the -RKRRKR- linker (SEQ ID NO: 41)Light Chain   1ANSFL                                     F WNKYKDGDQC ETSPCQNQGK  61CKDGLGEYTC TCLEGFEGKN CELFTRKLCS LDNGDCDQFC HEEQNSVVCS CARGYTLADN 121GKACIPTGPY PCGKQTLER Linker RKRRKR Heavy Chain 181               IVGGQE CKDGECPWQA LLINEENEGF CGGTILSEFY ILTAAHCLYQ 241AKRFKVRVGD RNTEQEEGGE AVHEVEVVIK HNRFTKETYD FDIAVLRLKT PITFRMNVAP 301ACLPERDWAE STLMTQKTGI VSGFGRTHEK GRQSTRLKML EVPYVDRNSC KLSSSFIITQ 361NMFCAGYDTK QEDACQGDAG GPHVTRFKDT YFVTGIVSWG EGCARKGKYG IYTKVTAFLK 421WIDRSMKTRG LPKAKSHAPE VITSSPLK

TABLE 13Sequence ID NO. 13 - Polypeptide Sequence of a Human Factor Xa triplemutant after removal of the -RKRRKR- linker (SEQ ID NO: 41) Light Chain  1 ANSFL                                     F WNKYKDGDQC ETSPCQNQGK 61 CKDGLGEYTC TCLEGFEGKN CELFTRKLCS LDNGDCDQFC HEEQNSVVCS CARGYTLADN121 GKACIPTGPY PCGKQTLER Heavy Chain 181               IVGGQE CKDGECPWQA LLINEENEGF CGGTILSEFY ILTAAHCLYQ 241AKRFKVRVGD RNTEQEEGGE AVHEVEVVIK HNRFTKETYD FDIAVLRLKT PITFRMNVAP 301ACLPERDWAE STLMTQKTGI VSGFGRTHEK GRQSTRLKML EVPYVDRNSC KLSSSFIITQ 361NMFCAGYDTK QEDACQGDAG GPHVTRFKDT YFVTGIVSWG EGCARKGKYG IYTKVTAFLK 421WIDRSMKTRG LPKAKSHAPE VITSSPLK

TABLE 14 Sequence ID NO. 14 - Polypeptide Sequence of Light ChainFragment of Human Factor Xa triple mutant after secretion 1ANSFL                                     F WNKYKDGDQC ETSPCQNQGK 61CKDGLGEYTC TCLEGFEGKN CELFTRKLCS LDNGDCDQFC HEEQNSVVCS CARGYTLADN 121GKACIPTGPY PCGKQTLER

TABLE 15 Sequence ID NO. 15 - Polypeptide Sequence of Heavy ChainFragment of Human Factor Xa triple mutant after secretion Heavy Chain181                IVGGQE CKDGECPWQA LLINEENEGF CGGTILSEFY ILTAAHCLYQ241 AKRFKVRVGD RNTEQEEGGE AVHEVEVVIK HNRFTKETYD FDIAVLRLKT PITFRMNVAP301 ACLPERDWAE STLMTQKTGI VSGFGRTHEK GRQSTRLKML EVPYVDRNSC KLSSSFIITQ361 NMFCAGYDTK QEDACQGDAG GPHVTRFKDT YFVTGIVSWG EGCARKGKYG IYTKVTAFLK421 WIDRSMKTRG LPKAKSHAPE VITSSPLK

TABLE 16 Sequence ID NO. 16 - A polynucleotide Sequence Encodingr-Antidote (a Factor X triple mutant) 1ATGGGGCGCC CACTGCACCT CGTCCTGCTC AGTGCCTCCC TGGCTGGCCT CCTGCTGCTC GGGGAAAGTC TGTTCATCCG CAGGGAGCAG GCCAACAACA 101TCCTGGCGAG GGTCACGAGG GCCAATTCCT TTCTTTTCTG GAATAAATAC AAAGATGGCG ACCAGTGTGA GACCAGTCCT TGCCAGAACC AGGGCAAATG 201TAAAGACGGC CTCGGGGAAT ACACCTGCAC CTGTTTAGAA GGATTCGAAG GCAAAAACTG TGAATTATTC ACACGGAAGC TCTGCAGCCT GGACAACGGG 301GACTGTGACC AGTTCTGCCA CGAGGAACAG AACTCTGTGG TGTGCTCCTG CGCCCGCGGG TACACCCTGG CTGACAACGG CAAGGCCTGC ATTCCCACAG 401GGCCCTACCC CTGTGGGAAA CAGACCCTGG AACGCAGGAA GAGGAGGAAG AGGATCGTGG GAGGCCAGGA ATGCAAGGAC GGGGAGTGTC CCTGGCAGGC 501CCTGCTCATC AATGAGGAAA ACGAGGGTTT CTGTGGTGGA ACCATTCTGA GCGAGTTCTA CATCCTAACG GCAGCCCACT GTCTCTACCA AGCCAAGAGA 601TTCAAGGTGA GGGTAGGGGA CCGGAACACG GAGCAGGAGG AGGGCGGTGA GGCGGTGCAC GAGGTGGAGG TGGTCATCAA GCACAACCGG TTCACAAAGG 701AGACCTATGA CTTCGACATC GCCGTGCTCC GGCTCAAGAC CCCCATCACC TTCCGCATGA ACGTGGCGCC TGCCTGCCTC CCCGAGCGTG ACTGGGCCGA 801GTCCACGCTG ATGACGCAGA AGACGGGGAT TGTGAGCGGC TTCGGGCGCA CCCACGAGAA GGGCCGGCAG TCCACCAGGC TCAAGATGCT GGAGGTGCCC 901TACGTGGACC GCAACAGCTG CAAGCTGTCC AGCAGCTTCA TCATCACCCA GAACATGTTC TGTGCCGGCT ACGACACCAA GCAGGAGGAT GCCTGCCAGG 1001GGGACGCAGG GGGCCCGCAC GTCACCCGCT TCAAGGACAC CTACTTCGTG ACAGGCATCG TCAGCTGGGG AGAGGGCTGT GCCCGTAAGG GGAAGTACGG 1101GATCTACACC AAGGTCACCG CCTTCCTCAA GTGGATCGAC AGGTCCATGA AAACCAGGGG CTTGCCCAAG GCCAAGAGCC ATGCCCCGGA GGTCATAACG 1201 TCCTCTCCAT TAAAGTGA

TABLE 17 Sequence ID. NO. 17- Polynucleotide Sequence of the r-Antidote Expression Vector 1TCTAGACACA GTACTCGGCC ACACCATGGG GCGCCCACTG CACCTCGTCC TGCTCAGTGC CTCCCTGGCT GGCCTCCTGC TGCTCGGGGA AAGTCTGTTC 101ATCCGCAGGG AGCAGGCCAA CAACATCCTG GCGAGGGTCA CGAGGGCCAA TTCCTTTCTT TTCTGGAATA AATACAAAGA TGGCGACCAG TGTGAGACCA 201GTCCTTGCCA GAACCAGGGC AAATGTAAAG ACGGCCTCGG GGAATACACC TGCACCTGTT TAGAAGGATT CGAAGGCAAA AACTGTGAAT TATTCACACG 301GAAGCTCTGC AGCCTGGACA ACGGGGACTG TGACCAGTTC TGCCACGAGG AACAGAACTC TGTGGTGTGC TCCTGCGCCC GCGGGTACAC CCTGGCTGAC 401AACGGCAAGG CCTGCATTCC CACAGGGCCC TACCCCTGTG GGAAACAGAC CCTGGAACGC AGGAAGAGGA GGAAGAGGAT CGTGGGAGGC CAGGAATGCA 501AGGACGGGGA GTGTCCCTGG CAGGCCCTGC TCATCAATGA GGAAAACGAG GGTTTCTGTG GTGGAACCAT TCTGAGCGAG TTCTACATCC TAACGGCAGC 601CCACTGTCTC TACCAAGCCA AGAGATTCAA GGTGAGGGTA GGGGACCGGA ACACGGAGCA GGAGGAGGGC GGTGAGGCGG TGCACGAGGT GGAGGTGGTC 701ATCAAGCACA ACCGGTTCAC AAAGGAGACC TATGACTTCG ACATCGCCGT GCTCCGGCTC AAGACCCCCA TCACCTTCCG CATGAACGTG GCGCCTGCCT 801GCCTCCCCGA GCGTGACTGG GCCGAGTCCA CGCTGATGAC GCAGAAGACG GGGATTGTGA GCGGCTTCGG GCGCACCCAC GAGAAGGGCC GGCAGTCCAC 901CAGGCTCAAG ATGCTGGAGG TGCCCTACGT GGACCGCAAC AGCTGCAAGC TGTCCAGCAG CTTCATCATC ACCCAGAACA TGTTCTGTGC CGGCTACGAC 1001ACCAAGCAGG AGGATGCCTG CCAGGGGGAC GCAGGGGGCC CGCACGTCAC CCGCTTCAAG GACACCTACT TCGTGACAGG CATCGTCAGC TGGGGAGAGG 1101GCTGTGCCCG TAAGGGGAAG TACGGGATCT ACACCAAGGT CACCGCCTTC CTCAAGTGGA TCGACAGGTC CATGAAAACC AGGGGCTTGC CCAAGGCCAA 1201GAGCCATGCC CCGGAGGTCA TAACGTCCTC TCCATTAAAG TGAGATCCCA CTCGGATCCC TATTCTATAG TGTCACCTAA ATGCTAGAGC TCGCTGATCA 1301GCCTCGACTG TGCCTTCTAG TTGCCAGCCA TCTGTTGTTT GCCCCTCCCC CGTGCCTTCC TTGACCCTGG AAGGTGCCAC TCCCACTGTC CTTTCCTAAT 1401AAAATGAGGA AATTGCATCG CATTGTCTGA GTAGGTGTCA TTCTATTCTG GGGGGTGGGG TGGGGCAGGA CAGCAAGGGG GAGGATTGGG AAGACAATAG 1501CAGGCATGCT GGGGATGCGG TGGGCTCTAT GGCTTCTGAG GCGGAAAGAA CCAGCTGGGG CTCGAGCGGC CGCCCCTTCT GAGGCGGAAA GAACCAGCTG 1601TGGAATGTGT GTCAGTTAGG GTGTGGAAAG TCCCCAGGCT CCCCAGCAGG CAGAAGTATG CAAAGCATGC ATCTCAATTA GTCAGCAACC AGGTGTGGAA 1701AGTCCCCAGG CTCCCCAGCA GGCAGAAGTA TGCAAAGCAT GCATCTCAAT TAGTCAGCAA CCATAGTCCC GCCCCTAACT CCGCCCATCC CGCCCCTAAC 1801TCCGCCCAGT TCCGCCCATT CTCCGCCCCA TGGCTGACTA ATTTTTTTTA TTTATGCAGA GGCCGAGGCC GCCTCGGCCT CTGAGCTATT CCAGAAGTAG 1901TGAGGAGGCT TTTTTGGAGG CCTAGGCTTT TGCAAAAAAG CTAGCTTCCC GCTGCCATCA TGGTTCGACC ATTGAACTGC ATCGTCGCCG TGTCCCAAAA 2001TATGGGGATT GGCAAGAACG GAGACCTACC CTGGCCTCCG CTCAGGAACG AGTTCAAGTA CTTCCAAAGA ATGACCACAA CCTCTTCAGT GGAAGGTAAA 2101CAGAATCTGG TGATTATGGG TAGGAAAACC TGGTTCTCCA TTCCTGAGAA GAATCGACCT TTAAAGGACA GAATTAATAT AGTTCTCAGT AGAGAACTCA 2201AAGAACCACC ACGAGGAGCT CATTTTCTTG CCAAAAGTTT GGATGATGCC TTAAGACTTA TTGAACAACC GGAATTGGCA AGTAAAGTAG ACATGGTTTG 2301GATAGTCGGA GGCAGTTCTG TTTACCAGGA AGCCATGAAT CAACCAGGCC ACCTTAGACT CTTTGTGACA AGGATCATGC AGGAATTTGA AAGTGACACG 2401TTTTTCCCAG AAATTGATTT GGGGAAATAT AAACTTCTCC CAGAATACCC AGGCGTCCTC TCTGAGGTCC AGGAGGAAAA AGGCATCAAG TATAAGTTTG 2501AAGTCTACGA GAAGAAAGAC TAACAGGAAG ATGCTTTCAA GTTCTCTGCT CCCCTCCTAA AGCTATGCAT TTTTATAAGA CCATGGGACT TTTGCTGGCT 2601TTAGATCCCG CGGAGATCCA GACATGATAA GATACATTGA TGAGTTTGGA CAAACCACAA CTAGAATGCA GTGAAAAAAA TGCTTTATTT GTGAAATTTG 2701TGATGCTATT GCTTTATTTG TAACCATTAT AAGCTGCAAT AAACAAGTTA ACAACAACAA TTGCATTCAT TTTATGTTTC AGGTTCAGGG GGAGGTGTGG 2801GAGGTTTTTT AAAGCAAGTA AAACCTCTAC AAATGTGGTA TGGCTGATTA TGAGCTCCAG CTTTTGTTCC CTTTAGTGAG GGTTAATTGC GCGCTTGGCG 2901TAATCATGGT CATAGCTGTT TCCTGTGTGA AATTGTTATC CGCTCACAAT TCCACACAAC ATACGAGCCG GAAGCATAAA GTGTAAAGCC TGGGGTGCCT 3001AATGAGTGAG CTAACTCACA TTAATTGCGT TGCGCTCACT GCCCGCTTTC CAGTCGGGAA ACCTGTCGTG CCAGCTGCAT TAATGAATCG GCCAACGCGC 3101GGGGAGAGGC GGTTTGCGTA TTGGGCGCTC TTCCGCTTCC TCGCTCACTG ACTCGCTGCG CTCGGTCGTT CGGCTGCGGC GAGCGGTATC AGCTCACTCA 3201AAGGCGGTAA TACGGTTATC CACAGAATCA GGGGATAACG CAGGAAAGAA CATGTGAGCA AAAGGCCAGC AAAAGGCCAG GAACCGTAAA AAGGCCGCGT 3301TGCTGGCGTT TTTCCATAGG CTCCGCCCCC CTGACGAGCA TCACAAAAAT CGACGCTCAA GTCAGAGGTG GCGAAACCCG ACAGGACTAT AAAGATACCA 3401GGCGTTTCCC CCTGGAAGCT CCCTCGTGCG CTCTCCTGTT CCGACCCTGC CGCTTACCGG ATACCTGTCC GCCTTTCTCC CTTCGGGAAG CGTGGCGCTT 3501TCTCATAGCT CACGCTGTAG GTATCTCAGT TCGGTGTAGG TCGTTCGCTC CAAGCTGGGC TGTGTGCACG AACCCCCCGT TCAGCCCGAC CGCTGCGCCT 3601TATCCGGTAA CTATCGTCTT GAGTCCAACC CGGTAAGACA CGACTTATCG CCACTGGCAG CAGCCACTGG TAACAGGATT AGCAGAGCGA GGTATGTAGG 3701CGGTGCTACA GAGTTCTTGA AGTGGTGGCC TAACTACGGC TACACTAGAA GGACAGTATT TGGTATCTGC GCTCTGCTGA AGCCAGTTAC CTTCGGAAAA 3801AGAGTTGGTA GCTCTTGATC CGGCAAACAA ACCACCGCTG GTAGCGGTGG TTTTTTTGTT TGCAAGCAGC AGATTACGCG CAGAAAAAAA GGATCTCAAG 3901AAGATCCTTT GATCTTTTCT ACGGGGTCTG ACGCTCAGTG GAACGAAAAC TCACGTTAAG GGATTTTGGT CATGAGATTA TCAAAAAGGA TCTTCACCTA 4001GATCCTTTTA AATTAAAAAT GAAGTTTTAA ATCAATCTAA AGTATATATG AGTAAACTTG GTCTGACAGT TACCAATGCT TAATCAGTGA GGCACCTATC 4101TCAGCGATCT GTCTATTTCG TTCATCCATA GTTGCCTGAC TCCCCGTCGT GTAGATAACT ACGATACGGG AGGGCTTACC ATCTGGCCCC AGTGCTGCAA 4201TGATACCGCG AGACCCACGC TCACCGGCTC CAGATTTATC AGCAATAAAC CAGCCAGCCG GAAGGGCCGA GCGCAGAAGT GGTCCTGCAA CTTTATCCGC 4301CTCCATCCAG TCTATTAATT GTTGCCGGGA AGCTAGAGTA AGTAGTTCGC CAGTTAATAG TTTGCGCAAC GTTGTTGCCA TTGCTACAGG CATCGTGGTG 4401TCACGCTCGT CGTTTGGTAT GGCTTCATTC AGCTCCGGTT CCCAACGATC AAGGCGAGTT ACATGATCCC CCATGTTGTG CAAAAAAGCG GTTAGCTCCT 4501TCGGTCCTCC GATCGTTGTC AGAAGTAAGT TGGCCGCAGT GTTATCACTC ATGGTTATGG CAGCACTGCA TAATTCTCTT ACTGTCATGC CATCCGTAAG 4601ATGCTTTTCT GTGACTGGTG AGTACTCAAC CAAGTCATTC TGAGAATAGT GTATGCGGCG ACCGAGTTGC TCTTGCCCGG CGTCAATACG GGATAATACC 4701GCGCCACATA GCAGAACTTT AAAAGTGCTC ATCATTGGAA AACGTTCTTC GGGGCGAAAA CTCTCAAGGA TCTTACCGCT GTTGAGATCC AGTTCGATGT 4801AACCCACTCG TGCACCCAAC TGATCTTCAG CATCTTTTAC TTTCACCAGC GTTTCTGGGT GAGCAAAAAC AGGAAGGCAA AATGCCGCAA AAAAGGGAAT 4901AAGGGCGACA CGGAAATGTT GAATACTCAT ACTCTTCCTT TTTCAATATT ATTGAAGCAT TTATCAGGGT TATTGTCTCA TGAGCGGATA CATATTTGAA 5001TGTATTTAGA AAAATAAACA AATAGGGGTT CCGCGCACAT TTCCCCGAAA AGTGCCACCT GGGAAATTGT AAACGTTAAT ATTTTGTTAA AATTCGCGTT 5101AAATTTTTGT TAAATCAGCT CATTTTTTAA CCAATAGGCC GAAATCGGCA AAATCCCTTA TAAATCAAAA GAATAGACCG AGATAGGGTT GAGTGTTGTT 5201CCAGTTTGGA ACAAGAGTCC ACTATTAAAG AACGTGGACT CCAACGTCAA AGGGCGAAAA ACCGTCTATC AGGGCGATGG CCCACTACGT GAACCATCAC 5301CCTAATCAAG TTTTTTGGGG TCGAGGTGCC GTAAAGCACT AAATCGGAAC CCTAAAGGGA GCCCCCGATT TAGAGCTTGA CGGGGAAAGC CGGCGAACGT 5401GGCGAGAAAG GAAGGGAAGA AAGCGAAAGG AGCGGGCGCT AGGGCGCTGG CAAGTGTAGC GGTCACGCTG CGCGTAACCA CCACACCCGC CGCGCTTAAT 5501GCGCCGCTAC AGGGCGCGTC GCGCCATTCG CCATTCAGGC TGCGCAACTG TTGGGAAGGG CGATCGGTGC GGGCCTCTTC GCTATTACGC CAGCTGGCGA 5601AAGGGGGATG TGCTGCAAGG CGATTAAGTT GGGTAACGCC AGGGTTTTCC CAGTCACGAC GTTGTAAAAC GACGGCCAGT GAGCGCGCGT AATACGACTC 5701ACTATAGGGC GAATTGGAAT TAATTCGCTG GGCTGAGACC CGCAGAGGAA GACGCTCTAG GGATTTGTCC CGGACTAGCG AGATGGCAAG GCTGAGGACG 5801GGAGGCTGAT TGAGAGGCGA AGGTACACCC TAATCTCAAT ACAACCCTTG GAGCTAAGCC AGCAATGGTA GAGGGAAGAT TCTGCACGTC CCTTCCAGGC 5901GGCCTCCCCG TCACCACCCA CCCCAACCCG CCCCGACCGG AGCTGAGAGT AATTCATACA AAAGGACTCG CCCCTGCCTT GGGGAATCCC AGGGACCGTC 6001GTTAAACTCC CACTAACGTA GAACCCAGAG ATCGCTGCGT TCCCGCCCCC TCACCCGCCC GCTCTCGTCA TCACTGAGGT GGAGAAGAGC ATGCGTGAGG 6101CTCCGGTGCC CGTCAGTGGG CAGAGCGCAC ATCGCCCACA GTCCCCGAGA AGTTGGGGGG AGGGGTCGGC AATTGAACCG GTGCCTAGAG AAGGTGGCGC 6201GGGGTAAACT GGGAAAGTGA TGTCGTGTAC TGGCTCCGCC TTTTTCCCGA GGGTGGGGGA GAACCGTATA TAAGTGCAGT AGTCGCCGTG AACGTTCTTT 6301TTCGCAACGG GTTTGCCGCC AGAACACAGG TAAGTGCCGT GTGTGGTTCC CGCGGGCCTG GCCTCTTTAC GGGTTATGGC CCTTGCGTGC CTTGAATTAC 6401TTCCACGCCC CTGGCTGCAG TACGTGATTC TTGATCCCGA GCTTCGGGTT GAAAGTGGGT GGGAGAGTTC GAGGCCTTGC GCTTAAGGAG CCCCTTCGCC 6501TCGTGCTTGA GTTGAGGCCT GGCTTGGGCG CTGGGGCCGC CGCGTGCGAA TCTGGTGGCA CCTTCGCGCC TATCTCGCTG CTTTCGATAA GTCTCTAGCC 6601ATTTAAAATT TTTGATGACC TGCTGCGACG CTTTTTTTCT GGCAAGATAG TCTTGTAAAT GCGGGCCAAG ATCTGCACAC TGGTATTTCG GTTTTTGGGG 6701CCGCGGGCGG CGACGGGGCC CGTGCGTCCC AGCGCACATG TTCGGCGAGG CGGGGCCTGC GAGCGCGGCC ACCGAGAATC GGACGGGGGT AGTCTCAAGC 6801TGGCCGGCCT GCTCTGGTGC CTGGCCTCGC GCCGCCGTGT ATCGCCCCGC CCTGGGCGGC AAGGCTGGCC CGGTCGGCAC CAGTTGCGTG AGCGGAAAGA 6901TGGCCGCTTC CCGGCCCTGC TGCAGGGAGC TCAAAATGGA GGACGCGGCG CTCGGGAGAG CGGGCGGGTG AGTCACCCAC ACAAAGGAAA AGGGCCTTTC 7001CGTCCTCAGC CGTCGCTTCA TGTGACTCCA CGGAGTACCG GGCGCCGTCC AGGCACCTCG ATTAGTTCTC GAGCTTTTGG AGTACGTCGT CTTTAGGTTG 7101GGGGGAGGGG TTTTATGCGA TGGAGTTTCC CCACACTGAG TGGGTGGAGA CTGAAGTTAG GCCAGCTTGG CACTTGATGT AATTCTCCTT GGAATTTGCC 7201CTTTTTGAGT TTGGATCTTG GTTCATTCTC AAGCCTCAGA CAGTGGTTCA AAGTTTTTTT CTTCCATTTC AGGTGTCGTG AAAACTACCC CTAAAAGCCA 7301 AAT

TABLE 18 Sequence ID NO. 18 - Signal Sequence forSEQ ID NOs. 12-15 and 19-25 Signal Sequence SP1MGRPLHLVLL SASLAGLLLL GESLFIRREQ ANNILARVTR

TABLE 19 Sequence ID NO. 19 - DesGla Xi (with activation peptide)with 2 RKR linkers (“RKR” disclosed as SEQ ID NO: 40 and “RKRRKR”disclosed as SEQ ID NO: 41) Light Chain   1ANSFL                                     F WNKYKDGDQC ETSPCQNQGK  61CKDGLGEYTC TCLEGFEGKN CELFTRKLCS LDNGDCDQFC HEEQNSVVCS CARGYTLADN 121GKACIPTGPY PCGKQTLER Linker RKRRKR Heavy Chain 121                        SVAQATSS SGEAPDSITW KPYDAADLDP TENPFDLLDF 181NQTQPERGDN NLTRIVGGQE CKDGECPWQA LLINEENEGF CGGTILSEFY ILTAAHCLYQ 241AKRFKVRVGD RNTEQEEGGE AVHEVEVVIK HNRFTKETYD FDIAVLRLKT PITFRMNVAP 301ACLPERDWAE STLMTQKTGI VSGFGRTHEK GRQSTRLKML EVPYVDRNSC KLSSSFIITQ 361NMFCAGYDTK QEDACQGDAG GPHVTRFKDT YFVTGIVSWG EGCARKGKYG IYTKVTAFLK 421WIDRSMKTRG LPKAKSHAPE VITSSPLK

TABLE 20 Sequence ID NO. 20 - DesGla Xai-beta form only-truncate at RLight Chain   1ANSFL                                     F WNKYKDGDQC ETSPCQNQGK  61CKDGLGEYTC TCLEGFEGKN CELFTRKLCS LDNGDCDQFC HEEQNSVVCS CARGYTLADN 121GKACIPTGPY PCGKQTLER Linker RKRRKR Heavy Chain 181               IVGGQE CKDGECPWQA LLINEENEGF CGGTILSEFY ILTAAHCLYQ 241AKRFKVRVGD RNTEQEEGGE AVHEVEVVIK HNRFTKETYD FDIAVLRLKT PITFRMNVAP 301ACLPERDWAE STLMTQKTGI VSGFGRTHEK GRQSTRLKML EVPYVDRNSC KLSSSFIITQ 361NMFCAGYDTK QEDACQGDAG GPHVTRFKDT YFVTGIVSWG EGCARKGKYG IYTKVTAFLK 421WIDRSMKTR

TABLE 21 Sequence ID NO. 21 - DesGlaXai alpha form only Light Chain   1ANSFL                                     F WNKYKDGDQC ETSPCQNQGK  61CKDGLGEYTC TCLEGFEGKN CELFTRKLCS LDNGDCDQFC HEEQNSVVCS CARGYTLADN 121GKACIPTGPY PCGKQTLER Linker RKRRKR Heavy Chain 181               IVGGQE CKDGECPWQA LLINEENEGF CGGTILSEFY ILTAAHCLYQ 241AKRFKVRVGD RNTEQEEGGE AVHEVEVVIK HNRFTKETYD FDIAVLRLKT PITFRMNVAP 301ACLPERDWAE STLMTQKTGI VSGFGRTHEK GRQSTRLKML EVPYVDRNSC KLSSSFIITQ 361NMFCAGYDTK QEDACQGDAG GPHVTRFKDT YFVTGIVSWG EGCARKGKYG IYTKVTAFLK 421WIDRSMKTQG LPKAKSHAPE VITSSPLK

TABLE 22 Sequence ID NO. 22 - DesGla Xai alpha form with glycosylationsite removed Light Chain   1ANSFL                                     F WNKYKDGDQC ETSPCQNQGK  61CKDGLGEYTC TCLEGFEGKN CELFTRKLCS LDNGDCDQFC HEEQNSVVCS CARGYTLADN 121GKACIPTGPY PCGKQTLER Linker RKRRKR Heavy Chain 181               IVGGQE CKDGECPWQA LLINEENEGF CGGTILSEFY ILTAAHCLYQ 241AKRFKVRVGD RNTEQEEGGE AVHEVEVVIK HNRFTKETYD FDIAVLRLKT PITFRMNVAP 301ACLPERDWAE STLMTQKTGI VSGFGRTHEK GRQSTRLKML EVPYVDRNSC KLSSSFIITQ 361NMFCAGYDTK QEDACQGDAG GPHVTRFKDT YFVTGIVSWG EGCARKGKYG IYTKVTAFLK 421WIDRSMKTQG LPKAKSHAPE VIASSPLK

TABLE 23 Sequence ID NO. 23 - DesGla δEGF1 Xai Light Chain   1 ANSFL  61                          TRKLCS LDNGDCDQFC HEEQNSVVCS CARGYTLADN 121GKACIPTGPY PCGKQTLER Linker RKRRKR Heavy Chain 181               IVGGQE CKDGECPWQA LLINEENEGF CGGTILSEFY ILTAAHCLYQ 241AKRFKVRVGD RNTEQEEGGE AVHEVEVVIK HNRFTKETYD FDIAVLRLKT PITFRMNVAP 301ACLPERDWAE STLMTQKTGI VSGFGRTHEK GRQSTRLKML EVPYVDRNSC KLSSSFIITQ 361NMFCAGYDTK QEDACQGDAG GPHVTRFKDT YFVTGIVSWG EGCARKGKYG IYTKVTAFLK 421WIDRSMKTRG LPKAKSHAPE VITSSPLK

TABLE 24 Sequence ID NO. 24 - DesGla Xi-RKR-alpha only form (“RKR”disclosed as SEQ ID NO: 40) Light Chain   1ANSFL                                     F WNKYKDGDQC ETSPCQNQGK  61CKDGLGEYTC TCLEGFEGKN CELFTRKLCS LDNGDCDQFC HEEQNSVVCS CARGYTLADN 121GKACIPTGPY PCGKQTLER Linker RKR Heavy Chain 121                        SVAQATSS SGEAPDSITW KPYDAADLDP TENPFDLLDF 181NQTQPERGDN NLTRIVGGQE CKDGECPWQA LLINEENEGF CGGTILSEFY ILTAAHCLYQ 241AKRFKVRVGD RNTEQEEGGE AVHEVEVVIK HNRFTKETYD FDIAVLRLKT PITFRMNVAP 301ACLPERDWAE STLMTQKTGI VSGFGRTHEK GRQSTRLKML EVPYVDRNSC KLSSSFIITQ 361NMFCAGYDTK QEDACQGDAG GPHVTRFKDT YFVTGIVSWG EGCARKGKYG IYTKVTAFLK 421WIDRSMKTQG LPKAKSHAPE VITSSPLK

TABLE 25 Sequence ID NO. 25 - triple mutant factor Xa construct withsingle RKR linker (SEQ ID NO: 40) Light Chain   1ANSFL                                     F WNKYKDGDQC ETSPCQNQGK  61CKDGLGEYTC TCLEGFEGKN CELFTRKLCS LDNGDCDQFC HEEQNSVVCS CARGYTLADN 121GKACIPTGPY PCGKQTLER Linker RKR Heavy Chain 181               IVGGQE CKDGECPWQA LLINEENEGF CGGTILSEFY ILTAAHCLYQ 241AKRFKVRVGD RNTEQEEGGE AVHEVEVVIK HNRFTKETYD FDIAVLRLKT PITFRMNVAP 301ACLPERDWAE STLMTQKTGI VSGFGRTHEK GRQSTRLKML EVPYVDRNSC KLSSSFIITQ 361NMFCAGYDTK QEDACQGDAG GPHVTRFKDT YFVTGIVSWG EGCARKGKYG IYTKVTAFLK 421WIDRSMKTRG LPKAKSHAPE VITSSPLK

TABLE 26Sequence ID NO. 26 - Des Gla Xai with the prepropeptide of humanprothrombin fused to ANSFL (SEQ ID NO: 42) Signal Sequence SP1MAHVRGLQLP GCLALAALCS LVHS Light Chain   1ANSFL                                     F WNKYKDGDQC ETSPCQNQGK  61CKDGLGEYTC TCLEGFEGKN CELFTRKLCS LDNGDCDQFC HEEQNSVVCS CARGYTLADN 121GKACIPTGPY PCGKQTLER Linker RKRRKR Heavy Chain 181               IVGGQE CKDGECPWQA LLINEENEGF CGGTILSEFY ILTAAHCLYQ 241AKRFKVRVGD RNTEQEEGGE AVHEVEVVIK HNRFTKETYD FDIAVLRLKT PITFRMNVAP 301ACLPERDWAE STLMTQKTGI VSGFGRTHEK GRQSTRLKML EVPYVDRNSC KLSSSFIITQ 361NMFCAGYDTK QEDACQGDAG GPHVTRFKDT YFVTGIVSWG EGCARKGKYG IYTKVTAFLK 421WIDRSMKTRG LPKAKSHAPE VITSSPLK

TABLE 27 Sequence ID NO. 27 - Alternate signal sequences, Des Gla Xaiwith transferrin signal peptide replacing native Xa signal peptideSignal Sequence SP1 MRLAVGALLV CAVLGLCLA Light Chain   1ANSFL                                     F WNKYKDGDQC ETSPCQNQGK  61CKDGLGEYTC TCLEGFEGKN CELFTRKLCS LDNGDCDQFC HEEQNSVVCS CARGYTLADN Linker121 GKACIPTGPY PCGKQTLER RKRRKR Heavy Chain 181               IVGGQE CKDGECPWQA LLINEENEGF CGGTILSEFY ILTAAHCLYQ 241AKRFKVRVGD RNTEQEEGGE AVHEVEVVIK HNRFTKETYD FDIAVLRLKT PITFRMNVAP 301ACLPERDWAE STLMTQKTGI VSGFGRTHEK GRQSTRLKML EVPYVDRNSC KLSSSFIITQ 361NMFCAGYDTK QEDACQGDAG GPHVTRFKDT YFVTGIVSWG EGCARKGKYG IYTKVTAFLK 421WIDRSMKTRG LPKAKSHAPE VITSSPLK

TABLE 28 Oligonucleotide primers used to construct thepolynucleotide encoding the r-Antidote triple mutant. SEQ ID NameSequence NO.  1. FXF1 AGTCTCTAGACACAGTACTCGGCCACACCAT 28 GGGG  2. FXR1AGCTGGATCCGAGTGGGATCTCACTTTAATG 29 GAGAGG  3. FXF2GAGGCCAGGAATGCAAGGACGG 30  4. FXR2 CCGTCCTTGCATTCCTGGCCTC 31  5. FXR3TCATCAGCGTGGACTCGGCCCAGT 32  6. GLAF CCAATTCCTTTCTTTTCTGGAATAAATACAA 33AGATGGCGACC  7. GLAR TTCCAGAAAAGAAAGGAATTGGCCCTCGTGA 34 CCC  8. FXAFGGAAGAGGAGGAAGAGGATCGTGGGAGGCCA 35 GGAA  9. FXARCACGATCCTCTTCCTCCTCTTCCTGCGTTCC 36 AGGG 10. ALA419 FAGGGGGACGCAGGGGGCCCGCACGTCACCC 37 11. ALA419 RGGGTGACGTGCGGGCCCCTGCTTCCCCCT 38

1. An isolated polypeptide comprising the amino acid sequence of SEQ IDNO. 21 or an amino acid sequence having at least 95% sequence identityto SEQ ID NO. 21, wherein the polypeptide has (a) reduced procoagulantactivity compared to wild-type factor Xa, (b) is capable of binding to afactor Xa inhibitor, (c) does not assemble into a prothrombinase complexand (d) prevents cleavage of amino acid residues 347-365 from thepolypeptide.
 2. The polypeptide of claim 1, wherein the amino acidsequence that has at least 95% sequence identity to SEQ ID NO. 21comprises a substitution, as compared to SEQ ID NO: 3, at the amino acidresidue corresponding to Arg429 of SEQ ID NO:
 3. 3. The polypeptide ofclaim 2, wherein the substitution is Arg 429Gln.
 4. The polypeptide ofclaim 2, wherein the amino acid sequence that has at least 95% sequenceidentity to SEQ ID NO. 21 comprises a substitution, as compared to SEQID NO: 3, at the amino acid residue corresponding to Thr443 of SEQ IDNO:
 3. 5. The polypeptide of claim 4, wherein the substitution isThr443Ala.
 6. The polypeptide of claim 1, wherein the amino acidsequence that has at least 95% sequence identity to SEQ ID NO. 21comprises a substitution, as compared to SEQ ID NO: 3, at the amino acidresidue corresponding to Ser436 of SEQ ID NO:
 3. 7. An isolatedpolypeptide comprising the amino acid sequence of SEQ ID NO.
 21. 8. Anisolated polypeptide consisting essentially of the amino acid sequenceof SEQ ID NO.
 21. 9. An isolated two-chain polypeptide prepared from thepolypeptide of claim 2 or 3 through cleavage at a linker located atamino acid residues 106-111.
 10. A peptide conjugate comprising acarrier covalently or non-covalently linked to a polypeptide of claim 1or
 7. 11. The peptide conjugate of claim 10, wherein the carrier is aliposome, a micelle, a pharmaceutically acceptable polymer, or apharmaceutically acceptable carrier.
 12. A pharmaceutical compositioncomprising a carrier and a polypeptide of claim 1 or
 7. 13. Apolynucleotide encoding for a polypeptide of claim 1 or
 7. 14. A methodfor preparing a polypeptide of claim 1 or 7 comprising expressing apolynucleotide encoding the polypeptide in a prokaryotic or eukaryotichost cell.
 15. The method of claim 14, further comprising isolating thepolypeptide.
 16. An isolated prokaryotic or eukaryotic host cellcomprising a polynucleotide encoding the polypeptide of claim 1 or 7.17. A composition comprising a carrier and a prokaryotic or eukaryotichost cell of claim
 16. 18. A method of neutralizing or reversinganticoagulation in a subject undergoing anticoagulant therapy with afactor Xa inhibitor in need of cessation of anticoagulation or sufferingfrom bleeding, comprising administering to the subject an effectiveamount of a composition of claim
 12. 19. The method of claim 18, whereinthe factor Xa inhibitor is selected from the group consisting offondaparinux, idraparinux, biotinylated idraparinux, enoxaparin,fragmin, NAP-5, rNAPc2, tissue factor pathway inhibitor, DX-9065a,YM-60828, YM-150, apixaban, rivaroxaban, PD-348292, otamixaban,edoxaban, LY517717, GSK913893, razaxaban, low molecular weight heparin,betrixaban or a pharmaceutically acceptable salt thereof, andcombinations thereof.
 20. The method of claim 18, wherein the subject isexperiencing a clinical major bleeding event selected from the groupconsisting of hemorrhage, bleeding into vital organs, bleeding requiringre-operation or a new therapeutic procedure, and a bleeding index ofequal to or more than 2.0 with an associated overt bleeding.
 21. Themethod of claim 18, wherein the composition is administered prior to asurgery.
 22. A method for selectively binding and inhibiting anexogenously administered factor Xa inhibitor in a subject, comprisingadministering to the subject an effective amount of a composition ofclaim
 12. 23. The method of claim 22, wherein the factor Xa inhibitor isselected from the group consisting of fondaparinux, idraparinux,biotinylated idraparinux, enoxaparin, fragmin, NAP-5, rNAPc2, tissuefactor pathway inhibitor, DX-9065a, YM-60828, YM-150, apixaban,rivaroxaban, PD-348292, otamixaban, edoxaban, LY517717, GSK913893,razaxaban, low molecular weight heparin, betrixaban or apharmaceutically acceptable salt thereof, and combinations thereof. 24.The method of claim 22, wherein the subject is experiencing a clinicalmajor bleeding event selected from the group consisting of hemorrhage,bleeding into vital organs, bleeding requiring re-operation or a newtherapeutic procedure, and a bleeding index of equal to or more than 2.0with an associated overt bleeding.
 25. The method of claim 24, whereinthe composition is administered prior to a surgery.